PT1409467E - A n-acylsphingosine glucosyltransferase inhibitor - Google Patents
A n-acylsphingosine glucosyltransferase inhibitor Download PDFInfo
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Abstract
Description
ΕΡ 1 409 467/ΡΤΕΡ 1 409 467 / ΡΤ
DESCRIÇÃO "Inibidor de N-acil-esfingosina-glucosiltransferase"DESCRIPTION " N-acyl-sphingosine-glucosyltransferase inhibitor "
Antecedentes do inventoBACKGROUND OF THE INVENTION
Os glicosfingolípidos (GSLs) são uma classe de compostos de ocorrência natural que possuem uma multitude de funções biológicas, incluindo a capacidade de promover o crescimento celular, diferenciação celular, aderência entre células ou entre células e proteínas de matriz, ligação de microorganismos e vírus a células, e células de metástases tumorais. Os GSLs são derivados a partir de glucosilceramida (GlcCer), que é produzida a partir de ceramida e UDP-glucose pela enzima UDP-glucose: IV-acil-esf ingosina-glucosiltransf erase (GlcCer-sintase) . A estrutura da ceramida é mostrada em seguida:Glycosphingolipids (GSLs) are a class of naturally occurring compounds that have a multitude of biological functions, including the ability to promote cell growth, cell differentiation, adhesion between cells or between cells and matrix proteins, binding of microorganisms and viruses to cells, and tumor metastasis cells. GSLs are derived from glucosylceramide (GlcCer), which is produced from ceramide and UDP-glucose by the enzyme UDP-glucose: IV-acyl-sphingosine-glucosyltransferase (GlcCer-synthase). The structure of ceramide is shown below:
OO
OHOH
Ceramida A acumulação de GSLs foi ligada a um número de doenças, incluindo as doenças de Tay-Sachs, de Gaucher e de Fabry (ver, por exemplo, Patente U.S. No. 6051598) . Os GSLs foram também ligados a certos cancros. Por exemplo, constatou-se que certos GSLs ocorrem apenas em tumores ou a concentrações anormalmente elevadas em tumores; exercem marcadas acções estimuladoras ou inibidoras no crescimento de tumores quando adicionados a células tumorais em meios de cultura; e inibem o normal sistema imunitário do corpo quando extravasados por tumores para o fluido extracelular circundante. A composição de um GSL de tumor modifica-se à medida que os tumores se tornam crescentemente malignos e anticorpos para certos GSLs inibem o crescimento de tumores. 2 ΕΡ 1 409 467/ΡΤCeramide Accumulation of GSLs has been linked to a number of diseases, including Tay-Sachs, Gaucher and Fabry diseases (see, for example, U.S. Patent No. 6051598). GSLs have also been linked to certain cancers. For example, it has been found that certain GSLs occur only in tumors or at abnormally high concentrations in tumors; exert marked stimulatory or inhibitory actions on tumor growth when added to tumor cells in culture media; and inhibit the body's normal immune system when extravasated by tumors into the surrounding extracellular fluid. The composition of a tumor GSL is modified as the tumors become increasingly malignant and antibodies to certain GSLs inhibit tumor growth. 2 ΕΡ 1 409 467 / ΡΤ
Os compostos que inibem a GlcCer-sintase podem reduzir as concentrações de GSL e foi reportado que são úteis para o tratamento de um indivíduo com uma das doenças anteriormente mencionadas. Um número de inibidores potentes de GlcCer, aqui referidos como "compostos amino do tipo ceramida", são divulgados nas Patentes U.S. Nos. 6051598, 5952370, 5945442, 5916911 e 6030995. O termo "compostos do tipo ceramida" refere-se a análogos de ceramida em que: 1) o álcool primário é substituído por um grupo amino substituído; e 2) o grupo alcenilo é substituído por um grupo arilo, preferivelmente fenilo ou fenilo substituído. Os compostos N-desacilados correspondentes são referidos como "compostos do tipo esfingosina".Compounds which inhibit GlcCer-synthase may reduce GSL concentrations and have been reported to be useful for the treatment of a subject with one of the above-mentioned diseases. A number of potent GlcCer inhibitors, referred to herein as " ceramide-like amino compounds ", are disclosed in U.S. Patents Nos. 6051598, 5952370, 5945442, 5916911 and 6030995. The term " ceramide compounds " refers to ceramide analogues wherein: 1) the primary alcohol is replaced by a substituted amino group; and 2) the alkenyl group is substituted by an aryl group, preferably phenyl or substituted phenyl. The corresponding N-deacylated compounds are referred to as " sphingosine type compounds ".
Infelizmente, os processos conhecidos para preparação de compostos amino do tipo ceramida são pouco adequados para o fabrico à escala industrial. Devido aos dois centros quirais, a maior parte das sínteses conhecidas gera quatro diastereoisómeros, o que resulta na necessidade de separar os diastereoisómeros por cromatografia e isolar o desejado enantiómero por cristalização após derivatização com reagente opticamente activos, e.g., isómeros de ácido dibenzoíl-tartárico (ver, por exemplo, Inokuchi e Radin, Journal of Lipid Research 28:565 (1987)). Nenhum dos processos é condutível em preparações em grande escala. A síntese enantiosselectiva de compostos amino do tipo ceramida utilizando reduções diastereosselectivas foi reportada (Mitchell, et al., J. Org. Chem. 63:8837 (1998) e Nishida, et al., SYNLETT 1998 :389 (1998)), mas requer mais de dez passos, alguns dos quais utilizam reagentes caros tais como hidreto de diisobutilalumínio (DIABAL) e aldeído de Garner ((R)-( + )-4-formil-2,2-dimetil-3-oxazolidinacarboxilato de tert-butilo). Assim, existe uma necessidade crítica de uma síntese enantiosselectiva de compostos amino do tipo ceramida que seja mais económica e eficiente, e envolva um menor número de passos do que as sínteses conhecidas.Unfortunately, known processes for preparing amino compounds of the ceramide type are poorly suited for industrial scale manufacture. Due to the two chiral centers, most of the known syntheses generate four diastereoisomers, which results in the need to separate the diastereomers by chromatography and to isolate the desired enantiomer by crystallization after derivatization with optically active reagents, eg, dibenzoyl tartaric acid isomers ( see, for example, Inokuchi and Radin, Journal of Lipid Research 28: 565 (1987)). None of the processes is amenable to large-scale preparation. The enantioselective synthesis of ceramide-type amino compounds using diastereoselective reductions has been reported (Mitchell, et al., J. Org. Chem. 63: 8837 (1998) and Nishida, et al., SYNLETT 1998: 389 (1998)), but requires more than ten steps, some of which use expensive reagents such as diisobutylaluminum hydride (DIABAL) and Garner's tert-butyl ((R) - (+) - 4-formyl-2,2-dimethyl-3-oxazolidinecarboxylate ). Thus, there is a critical need for an enantioselective synthesis of ceramide-like amino compounds which is more economical and efficient, and involves fewer steps than the known syntheses.
Lee et al. (J. Biol. Chem., 247 (21), pp. 14662-14669 (1999)) divulgam inibidores específicos de glucosilceramida-sintase. Em particular, Lee et al. divulgam apenas compostos que possuem um grupo alcanoílo C16 (palmitoílo) no átomo de azoto da amida. 3 ΕΡ 1 409 467/ΡΤLee et al. (J. Biol. Chem., 247 (21), pp. 14662-14669 (1999)) disclose specific glucosylceramide synthase inhibitors. In particular, Lee et al. disclose only compounds having a C16 alkanoyl (palmitoyl) group on the nitrogen atom of the amide. 3 ΕΡ 1 409 467 / ΡΤ
Sumário do invento É aqui proporcionada uma síntese eficiente e altamente enantiosselectiva de compostos amino do tipo ceramida. Esta síntese de compostos amino do tipo ceramida envolve apenas cinco passos a partir de compostos conhecidos. Por exemplo, o composto comparativo do tipo ceramida designado como "Composto 5" na Figura 1 foi produzido num excesso enantiomérico de pelo menos 99,6% e com um rendimento global de 9% (ver Exemplo comparativos 1 e 2). São também divulgados novos intermediários preparados no decurso da síntese.SUMMARY OF THE INVENTION Here is provided an efficient and highly enantioselective synthesis of ceramide-like amino compounds. This synthesis of ceramide-like amino compounds involves only five steps from known compounds. For example, the comparative ceramide compound designated " Compound 5 " in Figure 1 was produced in an enantiomeric excess of at least 99.6% and in an overall yield of 9% (see Comparative Example 1 and 2). New intermediates prepared in the course of the synthesis are also disclosed.
Num primeiro aspecto, o invento refere-se a um composto de acordo com a reivindicação 1. Uma concretização adicional do composto é apresentada na reivindicação 2. 0 invento, noutros aspectos, proporciona as utilizações dos compostos tal como apresentadas nas reivindicações 3 a 9.In a first aspect, the invention relates to a compound according to claim 1. A further embodiment of the compound is set forth in claim 2. The invention in other aspects provides the uses of the compounds as set forth in claims 3 to 9.
Breve descrição das Figuras A Figura 1 é um esquema que mostra a síntese do Composto comparativo (5) do tipo ceramida, utilizando o processo aqui divulgado. A Figura 2 é um esquema que mostra a síntese do Composto comparativo (13) do tipo ceramida, utilizando os processos aqui divulgados. A Figura 3 mostra as estruturas dos Compostos (5)-(8).BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic showing the synthesis of comparative Ceramide Compound (5) using the process disclosed herein. Figure 2 is a schematic showing the synthesis of comparative Ceramide Compound (13) using the procedures disclosed herein. Figure 3 shows the structures of Compounds (5) - (8).
Descrição detalhada do inventoDetailed Description of the Invention
Exemplos das condições específicas para realizar as reacções aqui descritas são proporcionados nos Exemplos comparativos 1 e 2. O termo "enantiómero", tal como aqui utilizado, e fórmulas estruturais que ilustram um enantiómero pretendem incluir o enantiómero "puro", isento do seu isómero óptico, assim como misturas do enantiómero e seu isómero óptico em que o enantiómero está presente num excesso enantiomérico, 4 ΕΡ 1 409 467/ΡΤ e.g., de pelo menos 10%, 25%, 50%, 75%, 90%, 95%, 98%, ou 99% de excesso enantiomérico.Examples of the specific conditions for carrying out the reactions described herein are provided in Comparative Examples 1 and 2. The term " enantiomer " as used herein, and structural formulas illustrating an enantiomer are intended to include the " pure " enantiomer, free from its isomer, as well as mixtures of the enantiomer and its optical isomer wherein the enantiomer is present in an enantiomeric excess, eg, at least 10%, 25%, 50%, 75%, 90%, 95% %, 98%, or 99% enantiomeric excess.
Os compostos deste invento que possuem um grupo suficientemente acídico, suficientemente básico, ou ambos grupos funcionais, podem em conformidade reagir com qualquer um de um número de bases inorgânicas, e ácidos inorgânicos e orgânicos, para formar um sal. São proporcionados sais fisiologicamente aceitáveis. Os ácidos habitualmente empregues para formar sais de adição de ácido são ácidos inorgânicos tais como ácido clorídrico, ácido bromidrico, ácido iodídrico, ácido sulfúrico, ácido fosfórico, e ácidos orgânicos tais como ácido p-toluenossulfónico, ácido metanossulfónico, ácido oxálico, ácido p-bromofenilsulfónico, ácido carbónico, ácido succínico, ácido cítrico, ácido benzóico e ácido acético. Exemplos de tais sais incluem o sulfato, pirossulfato, bissulfato, sulfito, bissulfito, fosfato, mono-hidrogenofosfato, di-hidrogenofosfato, meta-fosfato, pirofosfato, cloreto, brometo, iodeto, acetato, propionato, decanoato, caprilato, acrilato, formato, isobutirato, caproato, heptanoato, propiolato, oxalato, malonato, succinato, suberato, sebacato, fumarato, maleato, butino-1,4-dioato, hexino-1,6-dioato, benzoato, clorobenzoato, metilbenzoato, dinitrobenzoato, hidroxibenzoato, metoxi-benzoato, ftalato, sulfonato, xilenossulfonato, fenilacetato, fenilpropionato, fenilbutirato, citrato, lactato, gama-hidroxibutirato, glicolato, tartarato, metanossulfonato, propanossulfonato, naftaleno-l-sulfonato, naftaleno-2-sulfonato e mandelato.Compounds of this invention having a sufficiently acidic, sufficiently basic, or both functional groups may accordingly react with any of a number of inorganic bases, and inorganic and organic acids, to form a salt. Physiologically acceptable salts are provided. Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p- bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid and acetic acid. Examples of such salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, benzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gammahydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate and mandelate.
Sais de adição de base incluem os derivados a partir de bases inorgânicas, tais como hidróxidos, carbonatos e bicarbonatos de amónio ou metais alcalinos ou alcalino-terrosos. Tais bases úteis na preparação dos sais deste invento incluem assim hidróxido de sódio, hidróxido de potássio, hidróxido de amónio e carbonato de potássio. 5 ΕΡ 1 409 467/ΡΤBase addition salts include those derived from inorganic bases, such as ammonium, alkali or alkaline earth metal hydroxides, carbonates and bicarbonates. Such bases useful in the preparation of the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide and potassium carbonate. 5 ΕΡ 1 409 467 / ΡΤ
ExemplificaçãoExemplification
Exemplo comparativo 1 - Preparação em pequena escala de compostos do tipo ceramidaComparative Example 1 - Small scale preparation of ceramide compounds
Intermediário 1 (l.R,35,55,8a5)-l,3-Bis-(2',3' -di-hidro-benzo [l,4]dioxin-6'-il)-5-fenil-tetra-hidro-oxazolo[4,3-c][1,4]oxazin-8-onaIntermediate 1 (1R, 35,55,8a, 5) -1,3-Bis- (2 ', 3'-dihydro-benzo [1,4] dioxin-6'-yl) -5-phenyl-tetrahydro oxazolo [4,3-c] [1,4] oxazin-8-one
A uma solução agitada de (5Sj-5-fenilmorfolin-2-ona (2,00 g, 11,3 mmol) (preparada tal como em Dellaria, J.F.; Santarsiero, B.D., J. Org. Chem., 1989, 54, 3916) e 1,4- benzodioxan-6-carboxaldeído (5,56 g, 33,9 mmol) em tolueno o (125 mL) adicionaram-se peneiros moleculares de 4A (aproximadamente 20 mL). A mistura foi aquecida a refluxo durante 72 horas, filtrada para ficar isenta de peneiros e concentrada. A goma âmbar resultante foi cromatografada instantaneamente sobre sílica (éter dietílico/hexano) para proporcionar um sólido amarelo pálido. Este material foi purificado adicionalmente por trituração com éter dietílico para proporcionar 1,89 g (34%) de produto como um sólido branco esponjoso: ΧΗ RMN (CDCI3) δ 7,31-7,17 (m, 5H) , 6,95- 6,79 (m, 5H), 5,32-5,27 (m, 2H), 4,43-4,28 (m, 2H), 4,24 (s, 4H), 4,18 (m, 4H), 4,16-4,08 (m, 2H) ppm.To a stirred solution of (5S) -5-phenylmorpholin-2-one (2.00 g, 11.3 mmol) (prepared as in Dellaria, JF Santarsiero, BD, J. Org. Chem., 1989, 54, 3916) and 1,4-benzodioxan-6-carboxaldehyde (5.56 g, 33.9 mmol) in toluene (125 mL) was added 4A molecular sieves (approximately 20 mL). The resulting amber gum was flash chromatographed on silica (diethyl ether / hexane) to provide a pale yellow solid This material was further purified by trituration with diethyl ether to give 1.89 g (34%) of product as a fluffy white solid: ¹H NMR (CDCl3) δ 7.31-7.17 (m, 5H), 6.95-6.79 (m, 5H), 5.32-5.30 (M, 2H), 4.43-4.28 (m, 2H), 4.24 (s, 4H), 4.18 (m, 4H), 4.16-4.08 (m, 2H) ppm.
Intermediário 2 (25, 3.R, 1" 5) -3 - (2' , 3' -Di-hidro-benzo [l,4]dioxin-6'-il)-3-hidroxi-2-(2"-hidroxi-l"-fenil-etilamino)-1-pirrolidin-l-il-propan-l-onaIntermediate 2 (25, 3R, 1α) -3- (2 ', 3'-Dihydro-benzo [1,4] dioxin-6'-yl) -3-hydroxy-2- (2 " phenyl) -ethylamino) -1-pyrrolidin-1-yl-propan-1-one
6 ΕΡ 1 409 467/ΡΤ A uma solução agitada do Intermediário 1 (1,80 g, 3,69 mmol) em clorofórmio (20 mL) adicionou-se pirrolidina (2,0 mL, 24 mmol). A solução foi agitada durante a noite e depois concentrada. A espuma incolor aderente resultante foi tomada em metanol (16 mL) e ácido cloridrico IN (4 mL) . A mistura foi levada a refluxo durante 1 hora, tratada com ácido cloridrico IN adicional (2 mL) e levada a refluxo durante outras 2 horas. A solução reaccional foi concentrada e o resíduo resultante repartido entre acetato de etilo e uma solução aquosa de bicarbonato de sódio. A camada orgânica foi seca (sulfato de sódio) e concentrada. A goma amarelo pálido resultante foi purificada por cromatografia instantânea sobre sílica gel (diclorometano/amoníaco metanólico 2N) para proporcionar 1,40 g (92%) de Intermediário 2 como um sólido espumoso incolor: XH RMN (CDCI3) δ 7,31-7,13 (m, 5H) , 6,93-6,70 (m, 3H), 4,47 (d, J= 8,5, 1H) , 4,18 (s, 4H) , 3,82 (t, J=5, 9, 1H) , 3,74 (d, J=6,0, 2H) , 3,06 (d, J= 8,5, 1H) , 3,06-2,97 (m, 1H), 2,92-2,83 (m, 1H), 1,97-1,87 (m, 1H), 1,45-1,15 (m, 4H) ppm.To a stirred solution of Intermediate 1 (1.80 g, 3.69 mmol) in chloroform (20 mL) was added pyrrolidine (2.0 mL, 24 mmol). The solution was stirred overnight and then concentrated. The resulting adhering colorless foam was taken up in methanol (16 mL) and 1N hydrochloric acid (4 mL). The mixture was refluxed for 1 hour, treated with 1N hydrochloric acid (2 mL) and refluxed for another 2 hours. The reaction solution was concentrated and the resulting residue partitioned between ethyl acetate and an aqueous solution of sodium bicarbonate. The organic layer was dried (sodium sulfate) and concentrated. The resulting pale yellow gum was purified by flash chromatography on silica gel (dichloromethane / 2N methanolic ammonia) to provide 1.40 g (92%) of Intermediate 2 as a colorless foamy solid: 1 H NMR (CDCl 3) δ 7.31-7 (M, 3H), 4.47 (d, J = 8.5, 1H), 4.18 (s, 4H), 3.82 (t, J = 5.9, 1H), 3.74 (d, J = 6.0, 2H), 3.06 (d, J = 8.5,1H), 3.06-2.97 (m, 1H), 2.92-2.83 (m, 1H), 1.97-1.87 (m, 1H), 1.45-1.15 (m, 4H) ppm.
Intermediário 3 (1-R, 2 R, 1"S) -1- (2' , 3' -Di-hidro-benzo [l,4]dioxin-6'-il)-2-(2"-hidroxi-l"-fenil-etilamino)-3-pirrolidin-l-il-propan-l-olIntermediate 3 (1R, 2R, 1S) -1- (2 ', 3'-Dihydro-benzo [1,4] dioxin-6'-yl) -2- (2'- phenylaminoethyl) -3-pyrrolidin-1-yl-propan-1-ol
A uma solução agitada do Intermediário 2 (1,38 g, 3,35 mmol) em tetra-hidrofurano (30 mL) adicionou-se hidreto de alumínio e lítio (0,26 g, 6,9 mmol). A suspensão espumosa foi agitada durante a noite e depois neutralizada com a adição (gota a gota até cessar a formação de espuma) de solução aquosa de hidróxido de sódio IN (13 mL) . A mistura foi diluída com água e extraída com acetato de etilo. A camada orgânica foi seca (sulfato de sódio) e concentrada para proporcionar uma goma incolor. Cromatografia instantânea sobre sílica gel (diclorometano/amoníaco metanólico 2N) 7 ΕΡ 1 409 467/ΡΤ proporcionou 0,94 g (70%) de produto como uma espuma aderente e incolor: XH RMN (CDCI3) δ 7,36-7, 17 (m, 5H) , 6,88- -6, 74 (m 3H), 4,42 (d, J=5, 4, 1H), 4,26 (s, , 4H), 3,79-3,69 (m, 1H) 3,64-3,56 (m, 1H), 3,55-3, 45 (m, 1H), 3,00-2,90 (m, 1H) 2,67-2,57 (m, 1H), 2,43-2,32 (m, 4H) , 2,25-2,15 (m, 1H) 1,75-1,65 (m, 4H) ppm.To a stirred solution of Intermediate 2 (1.38 g, 3.35 mmol) in tetrahydrofuran (30 mL) was added lithium aluminum hydride (0.26 g, 6.9 mmol). The foam suspension was stirred overnight and then neutralized by the addition (dropwise until frothing) of 1N aqueous sodium hydroxide solution (13 mL). The mixture was diluted with water and extracted with ethyl acetate. The organic layer was dried (sodium sulfate) and concentrated to provide a colorless gum. Flash chromatography on silica gel (dichloromethane / 2N methanolic ammonia) Î'1 409 467 / ΡΤ provided 0.94 g (70%) of product as a colorless, adherent foam: 1 H NMR (CDCl3) δ 7.36-7.17 (m, 5H), 6.88-6.74 (m 3H), 4.42 (d, J = 5.4, 1H), 4.26 (s, 4H), 3.79-3, (M, 1H), 3.55-3.45 (m, 1H), 3.00-2.90 (m, 1H), 2.67-2.68 (m, 57 (m, 1H), 2.43-2.32 (m, 4H), 2.25-2.15 (m, 1H), 1.75-1.65 (m, 4H) ppm.
Intermediário 4 (IR,2R)-2-Amino-l-{2',3'-di-hidro-benzo[1,4]dioxin-6'-il)-3-pirrolidin-l-il-propan-l-olIntermediate 4 (1R, 2R) -2-Amino-1- {2 ', 3'-dihydro-benzo [1,4] dioxin-6'-yl) -3-pyrrolidin-1-yl-propan-1 -ole
Numa bomba reaccional de elevada pressão equipada com um agitador mecânico carregou-se uma solução do Intermediário 3 (0,91 g, 2,28 mmol) em metanol/água 10:1 (22 mL) , ácido trifluoroacético (0,18 mL, 2,3 mmol) e hidróxido de paládio a 20% sobre carbono (catalisador de Perlman; 0,91 g). O reactor foi evacuado e cheio com árgon três vezes e depois evacuado e cheio com hidrogénio (100 psi) . A mistura reaccional foi agitada durante 2 dias e depois evacuada e soprada com azoto. A solução reaccional foi filtrada através de Celite e concentrada. A goma cinzento-esverdeado resultante foi sujeita a cromatografia instantânea sobre sílica gel (diclorometano/amoníaco metanólico 2N) para proporcionar 0,165 g (26%) de produto como uma goma quase incolor: 1H RMN (CDC13) δ 6, 89-6, 76 (m, 3H) , 4,54 (d, J=3, 7, 1H) , 4,25 (s, 4H) , 3,43 (s, 1H), 3,14-3,07 (m, 1H) , 2,68-2,41 (m, 6H) , 1,82-1,71 (m, 4H) ppm.Into a high pressure reaction pump equipped with a mechanical stirrer was charged a solution of Intermediate 3 (0.91 g, 2.28 mmol) in 10: 1 methanol / water (22 mL), trifluoroacetic acid (0.18 mL, 2.3 mmol) and 20% palladium hydroxide on carbon (Perlman's catalyst: 0.91 g). The reactor was evacuated and filled with argon three times and then evacuated and filled with hydrogen (100 psi). The reaction mixture was stirred for 2 days and then evacuated and blown with nitrogen. The reaction solution was filtered through Celite and concentrated. The resulting gray-green gum was flash chromatographed on silica gel (dichloromethane / 2N methanolic ammonia) to afford 0.165 g (26%) of product as an almost colorless gum: 1 H NMR (CDCl 3) δ 6.96-6.76 (m, 3H), 4.54 (d, J = 3.7, 1H), 4.25 (s, 4H), 3.43 (s, 1H), 3.14-3.07 (m, 1H ), 2.68-2.41 (m, 6H), 1.82-1.71 (m, 4H) ppm.
Composto 5 [2 - (2',3'-di-hidrobenzo[1,4]dioxin-6'-il)-2-hidroxi-l-pirrolidin-l-ilmetiletil]amida de ácido (IR,2R)-hexadecanóico ΕΡ 1 409 467/ΡΤCompound 5 (1R, 2R) -hexadecanoic acid [2- (2 ', 3'-dihydrobenzo [1,4] dioxin-6'-yl) -2-hydroxy-1-pyrrolidin-1-ylmethylethyl] ΕΡ 1 409 467 / ΡΤ
A uma solução agitada do Intermediário 4 (0,165 g, 0,593 mmol) em diclorometano (8 mL) juntou-se cloreto de palmitoílo (0,18 g, 0,59 mmol) seguido de N,N-diisopropiletilamina (0,11 mL, 0,65 mmol). A solução foi agitada durante 2 horas e depois concentrada. O resíduo foi repartido entre acetato de etilo e uma solução aquosa de bicarbonato de sódio. A camada orgânica foi seca (sulfato de sódio) e concentrada. O sólido esbranquiçado resultante foi sujeito a cromatografia instantânea sobre sílica gel (diclorometano/amoníaco metanólico 2N) para proporcionar 0,174 g (57%) de produto como um sólido branco. Comparações por espectroscopia de 1H RMN e HPLC quiral analítica (coluna: Chirex (S)-VAL e (R)-NE, 4,6x250 mm; eluente: ácido trifluoroacético a 0,5% em hexano/diclorometano/etanol 67:31:2; caudal: 1 mL/min;To a stirred solution of Intermediate 4 (0.165 g, 0.593 mmol) in dichloromethane (8 mL) was added palmitoyl chloride (0.18 g, 0.59 mmol) followed by N, N-diisopropylethylamine (0.11 mL, 0.65 mmol). The solution was stirred for 2 hours and then concentrated. The residue was partitioned between ethyl acetate and an aqueous solution of sodium bicarbonate. The organic layer was dried (sodium sulfate) and concentrated. The resulting off-white solid was flash chromatographed on silica gel (dichloromethane / 2N methanolic ammonia) to provide 0.174 g (57%) of product as a white solid. Comparisons by 1H NMR spectroscopy and analytical chiral HPLC (column: Chirex (S) -VAL and (R) -NE, 4.6x250 mm; eluent: 0.5% trifluoroacetic acid in hexane / dichloromethane / ethanol 67:31: 2, flow rate: 1 mL / min;
detecção 280 nM) demonstraram que este material era idêntico a uma amostra do mesmo composto preparado pelo processo de Polt, et al. (J. Org. Chem., 1998, 63, 8837). O excesso enantiomérico foi determinado como sendo de 99,6%. A contaminação total a partir dos dois diastereoisómeros possíveis foi determinada como sendo de 0,2%. ΧΗ RMN (CDCI3) δ 6, 88-6, 73 (m, 3H) , 5,84 (d, J= 7,3, 1H) , 4,90 (d, J= 3,8, 1H) , 4,24 (s, 4H) , 4,22-4,15 (m, 1H) , 2, 86-2, 72 (m, 2H) , 2,72-2,55 (m, 4H) , 2,10 (t, J=1,5, 2H) , 1, 82-1, 74 (m, 4H) , 1,58-1,46 (m, 2H), 1,32-1,16 (m, 24H), 0,88 (t, J=6,7, 3H) ppm.detection 280 nM) demonstrated that this material was identical to a sample of the same compound prepared by the method of Polt, et al. (J. Org. Chem., 1998, 63, 8837). The enantiomeric excess was determined to be 99.6%. The total contamination from the two possible diastereoisomers was determined to be 0.2%. Δ NMR (CDCl3) δ 6.88-6.73 (m, 3H), 5.84 (d, J = 7.3, 1H), 4.90 (d, J = 3.8, 1H), 4. (M, 1H), 2.86-2.72 (m, 2H), 2.72-2.55 (m, 4H), 2.10 (t, J = 1.5, 2H), 1.82-1.74 (m, 4H), 1.58-1.46 (m, 2H), 1.32-1.16 (m, 24H) , 0.88 (t, J = 6.7, 3H) ppm.
Exemplo comparativo 2 - Preparação em larga escala de compostos do tipo ceramida (5 S)-5-Fenilmorfolin-2-onaComparative Example 2 - Large-scale preparation of ceramide (5S) -5-Phenylmorpholin-2-one compounds
Ph. o ^ nh2 l OPhPh.
Ph. CHjCN -► ΌΗPh. CHjCN -► ΌΗ
Br H 1Br H 1
9 ΕΡ 1 409 467/ΡΤ9 Ε 1 409 467 / ΡΤ
Preparou-se uma solução de S-(+)-fenilglicinol (Aldrich, 10,17 g, 78,12 mmol) e diisopropiletilamina (Aldrich, 34 mL, 195 mmol, 2,5 equivalentes) em CH3CN (200 mL) . Esta solução foi adicionada a α-bromoacetato de fenilo (18,48 g, 85,9 mmol, 1,1 equivalentes) dissolvido em CH3CN (50 mL) sob azoto, gota a gota durante 2 horas. A solução resultante foi agitada sob azoto durante 16-20 horas. O solvente foi removido por roto-evaporação, mantendo a temperatura do banho inferior a 25°C. Ao óleo juntou-se acetato de etilo (120 mL) e a mistura foi agitada durante 15 minutos. O precipitado branco resultante foi removido por filtração e o sólido lavado com acetato de etilo (25 mL) . O filtrado foi roto-evaporado até ficar um óleo, mantendo a temperatura do banho inferior a 25°C. Após secagem sob vácuo durante 0,5 horas, o óleo foi dissolvido em CH2CI2 (17 mL) e carregado numa coluna de sílica gel (60 g cheia com acetato de etilo a 10%/hexanos). As manchas superiores de subprodutos foram eluidas com acetato de etilo a 10%/hexanos e o produto foi eluído com acetato de etilo a 50%/hexanos-100% de acetato de etilo. As fracções contendo o produto foram roto-evaporadas até ficar um óleo, mantendo a temperatura do banho inferior a 25°C. Este óleo foi dissolvido em acetato de etilo (12 mL) e juntaram-se hexanos (60 mL) lentamente num banho de gelo para precipitar o produto. 0 precipitado resultante foi filtrado. O sólido branco a amarelo foi seco sob vácuo. A (5S)-5-fenil-morfolin-2-ona obtida (7,4 g, 41,8 mmol, 53%) foi utilizada directamente no passo seguinte.A solution of S - (+) - phenylglycinol (Aldrich, 10.17 g, 78.12 mmol) and diisopropylethylamine (Aldrich, 34 mL, 195 mmol, 2.5 equivalents) in CH3 CN (200 mL) was prepared. This solution was added to phenyl α-bromoacetate (18.48 g, 85.9 mmol, 1.1 equivalents) dissolved in CH 3 CN (50 mL) under nitrogen, dropwise over 2 hours. The resulting solution was stirred under nitrogen for 16-20 hours. The solvent was removed by rotoevaporation, keeping the bath temperature below 25øC. To the oil was added ethyl acetate (120 mL) and the mixture was stirred for 15 minutes. The resulting white precipitate was removed by filtration and the solid washed with ethyl acetate (25 mL). The filtrate was rotoevaporated to an oil, keeping the bath temperature below 25 ° C. After drying under vacuum for 0.5 hours, the oil was dissolved in CH 2 Cl 2 (17 mL) and loaded onto a silica gel column (60 g filled with 10% ethyl acetate / hexanes). The upper byproduct stains were eluted with 10% ethyl acetate / hexanes and the product eluted with 50% ethyl acetate / hexanes-100% ethyl acetate. The fractions containing the product were rotoevaporated to an oil, keeping the bath temperature below 25 ° C. This oil was dissolved in ethyl acetate (12 mL) and hexanes (60 mL) were added slowly in an ice bath to precipitate the product. The resulting precipitate was filtered. The white to yellow solid was dried under vacuum. The (5S) -5-phenyl-morpholin-2-one obtained (7.4 g, 41.8 mmol, 53%) was used directly in the next step.
Intermediário 1 (1-R, 35, 55, 8a5) -1,3-Bis- (2' ,3' -di-hidrobenzo [l,4]dioxin-6'~ il)-5-feniltetra-hidro-oxazolo[4,3-c][1,4]oxazin-8-onaIntermediate 1 (1R, 35,55,8a) -1,3-Bis- (2 ', 3'-dihydrobenzo [1,4] dioxin-6'-yl) -5-phenyltetrahydro-oxazole [4,3-c] [1,4] oxazin-8-one
Dissolveram-se (5 S)-5-fenilmorfolin-2-ona (7,4 g, 41,8 mmol) e benzodioxolano-6-carboxaldeído (Aldrich ou Alfa 10 ΕΡ 1 409 467/ΡΤ(5 S) -5-Phenylmorpholin-2-one (7.4 g, 41.8 mmol) and benzodioxolane-6-carboxaldehyde (Aldrich or Alfa 10 ΕΡ 1 409 467 / Diss
Aesar, 20,56 g, 125,2 mmol, 3,0 equivalentes) em tolueno (180 mL) . A solução foi colocada num aparelho extractor soxhlet cheio de peneiros moleculares de 4 À (cerca de 30 g) . A solução foi levada a refluxo sob azoto durante 2-3 dias. Após arrefecimento até temperatura ambiente, o solvente foi removido por roto-evaporação e o óleo foi dissolvido em acetato de etilo (200 mL). Juntou-se uma solução de bissulfito de sódio (Aldrich, 50 g) em água (100 mL) e a mistura de duas fases foi agitada a temperatura ambiente durante 1 hora. O sólido branco resultante foi removido por filtração e lavado com acetato de etilo. O filtrado foi colocado num funil de separação e as camadas separadas. A camada orgânica foi lavada com água (100 mL) e solução saturada de cloreto de sódio (100 mL) . A solução seca (Na2S04) foi filtrada e roto-evaporada até ficar um óleo espumoso amarelo-vermelho (23,11 g) . Após secagem sob vácuo durante 1 hora, juntou-se éter dietilico (350 ml) e a mistura foi agitada a temperatura ambiente durante 16-20 horas. O sólido branco-amarelo resultante foi filtrado. O sólido foi seco sob vácuo. O cicloaducto foi obtido num rendimento de 46% (9,34 g) .Aesar, 20.56 g, 125.2 mmol, 3.0 equivalents) in toluene (180 mL). The solution was placed in a soxhlet extractor apparatus filled with 4Å molecular sieves (about 30 g). The solution was refluxed under nitrogen for 2-3 days. After cooling to room temperature, the solvent was removed by rotoevaporation and the oil was dissolved in ethyl acetate (200 mL). A solution of sodium bisulfite (Aldrich, 50 g) in water (100 ml) was added and the two phase mixture was stirred at room temperature for 1 hour. The resulting white solid was removed by filtration and washed with ethyl acetate. The filtrate was placed on a separatory funnel and the layers separated. The organic layer was washed with water (100 mL) and brine (100 mL). The dried solution (Na 2 SO 4) was filtered and roto-evaporated to a yellow-red foamy oil (23.11 g). After drying under vacuum for 1 hour, diethyl ether (350 ml) was added and the mixture was stirred at room temperature for 16-20 hours. The resulting white-yellow solid was filtered. The solid was dried under vacuum. The cycloadduct was obtained in 46% yield (9.34 g).
Intermediário 2 (2 5, 3.R, l"5)-3-(2',3' -Di-hidrobenzo [l,4]dioxin-6'-il)-3-hidroxi-2-(2"-hidroxi-l"-fenil-etilamino)-1-pirrolidin-l-il-propan-l-onaIntermediate 2 (2 S, 5 R, 1 R) -3- (2 ', 3'-Dihydrobenzo [1,4] dioxin-6'-yl) -3-hydroxy-2- (2 " hydroxy-1'-phenyl-ethylamino) -1-pyrrolidin-1-yl-propan-1-one
Ao cicloaducto (Intermediário 1, 6,7 g, 13,74 mmol) dissolvido em diclorometano (40 mL) juntou-se pirrolidina (Aldrich, 5,7 mL, 68,7 mmol, 5 equivalentes). A solução foi agitada sob azoto a temperatura ambiente durante 16-18 horas. O solvente foi roto-evaporado para proporcionar um óleo espumoso amarelo que foi seco sob vácuo durante 0,5 horas. 0 produto bruto foi dissolvido em metanol (115 mL) e juntou-se uma solução aquosa de HC1 1M (115 mL). A solução foi levada a refluxo durante 4 horas. Após arrefecimento a temperatura 11 ΕΡ 1 409 467/ΡΤ ambiente, ο metanol foi removido por roto-evaporação. Juntou-se acetato de etilo (60 mL) e o sistema de duas fases foi agitado a temperatura ambiente durante 5-15 minutos. As duas camadas foram separadas e a camada orgânica foi extraída com HC1 1M (30 mL) . As camadas aquosas combinadas foram lavadas com acetato de etilo (60, 30 mL) . Juntou-se lentamente uma solução saturada de bicarbonato de sódio (150 mL) à camada aquosa. O produto foi extraído três vezes com acetato de etilo (60 mL) a partir da camada aquosa básica (pH = 8-9). As camadas orgânicas combinadas contendo o produto foram lavadas com uma solução saturada de cloreto de sódio (30 mL) . Após secagem com Na2S04 a solução foi filtrada e roto-evaporada para proporcionar um sólido amarelo. O Intermediário 2 foi obtido num rendimento de 93% (5,26 g).To the cycloadduct (Intermediate 1, 6.7 g, 13.74 mmol) dissolved in dichloromethane (40 mL) was added pyrrolidine (Aldrich, 5.7 mL, 68.7 mmol, 5 equivalents). The solution was stirred under nitrogen at room temperature for 16-18 hours. The solvent was rotoevaporated to provide a yellow foamy oil which was dried under vacuum for 0.5 hours. The crude product was dissolved in methanol (115 mL) and 1M aqueous HCl (115 mL) was added. The solution was refluxed for 4 hours. After cooling to ambient temperature, the methanol was removed by roto-evaporation. Ethyl acetate (60 mL) was added and the two phase system was stirred at room temperature for 5-15 minutes. The two layers were separated and the organic layer was extracted with 1 M HCl (30 mL). The combined aqueous layers were washed with ethyl acetate (60, 30 mL). Saturated sodium bicarbonate solution (150 mL) was slowly added to the aqueous layer. The product was extracted three times with ethyl acetate (60 mL) from the basic aqueous layer (pH = 8-9). The combined organic layers containing the product were washed with brine (30 mL). After drying with Na 2 SO 4 the solution was filtered and roto-evaporated to give a yellow solid. Intermediate 2 was obtained in 93% yield (5.26 g).
Intermediário 3 (IA, 2.R, l"5j-l-(2',3' -Di-hidrobenzo [l,4]dioxin-6'-il)-2-(2-hidroxi-l"-fenil-etilamino)-3-pirrolidin-l-il-propan-l-olIntermediate 3 (1α, 2R, 1α) -11- (2 ', 3'-Dihydrobenzo [1,4] dioxin-6'-yl) -2- (2-hydroxy-1'- ethylamino) -3-pyrrolidin-1-yl-propan-1-ol
O OH OHOH OH
OH OH A um frasco de 3 tubuladuras equipado com funil de adição e condensador juntaram-se LiAlH4 (Aldrich, 1,2 g, 31,7 mmol, 2,5 equivalentes) e THF anidro (20 mL) sob azoto. Adicionou-se gota a gota à reacção durante 15-30 minutos uma solução de Intermediário 2 (5,23 g, 12,68 mmol) em THF anidro (75 mL) . A reacção foi levada a refluxo sob azoto durante 9 horas. A reacção foi arrefecida num banho de gelo e juntou-se cuidadosamente gota a gota uma solução de NaOH 1M. Após agitação a temperatura ambiente durante 15 minutos, juntaram-se água (50 mL) e acetato de etilo (75 mL) . As camadas foram separadas e a camada aquosa foi extraída duas vezes com acetato de etilo (75 mL) . As camadas orgânicas combinadas foram lavadas com uma solução saturada de cloreto de sódio (25 mL) . Após secagem com Na2S04, a solução foi filtrada e roto-evaporada para proporcionar um óleo espumoso de incolor 12 ΕΡ 1 409 467/ΡΤ a amarelo. Ο Intermediário 3 foi obtido num rendimento de 99% (5,3 g).OH To a 3-necked flask equipped with addition funnel and condenser were added LiAlH 4 (Aldrich, 1.2 g, 31.7 mmol, 2.5 equivalents) and anhydrous THF (20 mL) under nitrogen. A solution of Intermediate 2 (5.23 g, 12.68 mmol) in anhydrous THF (75 mL) was added dropwise to the reaction over 15-30 minutes. The reaction was refluxed under nitrogen for 9 hours. The reaction was cooled in an ice bath and 1M NaOH solution was carefully dropwise added. After stirring at room temperature for 15 minutes, water (50 mL) and ethyl acetate (75 mL) were added. The layers were separated and the aqueous layer extracted twice with ethyl acetate (75 mL). The combined organic layers were washed with brine (25 mL). After drying with Na 2 SO 4, the solution was filtered and roto-evaporated to provide a colorless foam oil 12 Ρ Ρ 1 409 467 / ΡΤ in yellow. Intermediate 3 was obtained in 99% yield (5.3 g).
Intermediário 4 (1R,2R)-2-Amino-l-(2',3'-di-hidrobenzo[1,4]dioxin-6'-il)-3-pirrolidin-l-il-propan-l-olIntermediate 4 (1R, 2R) -2-Amino-1- (2 ', 3'-dihydrobenzo [1,4] dioxin-6'-yl) -3-pyrrolidin-1-yl-propan-1-ol
0 Intermediário 3 (5,3 g, 13,3 mmol) foi dissolvido em metanol (60 mL) . Adicionaram-se água (6 mL) e ácido trifluoroacético (2,05 mL, 26,6 mmol, 2 equivalentes). Após ter sido colocado sob azoto, juntou-se hidróxido de paládio a 20% sobre carbono (catalisador de Pearlman, Lancaster ou Aldrich, 5,3 g) . A mistura foi colocada num dispositivo de Reactor Pressurizado Parr com inserção de vidro. O dispositivo foi colocado sob azoto e depois sob uma pressão de hidrogénio de 110-120 psi. A mistura foi agitada durante 2-3 dias a temperatura ambiente sob pressão de hidrogénio de 100-120 psi. A mistura reaccional foi colocada sob azoto e filtrada através de uma almofada de celite. A almofada de celite foi lavada com metanol (100 mL) e água (100 mL) . 0 metanol foi removido por roto-evaporação. A camada aquosa foi lavada com acetato de etilo três vezes (100, 50, 50 mL) .Intermediate 3 (5.3 g, 13.3 mmol) was dissolved in methanol (60 mL). Water (6 mL) and trifluoroacetic acid (2.05 mL, 26.6 mmol, 2 equivalents) were added. After being placed under nitrogen, 20% palladium hydroxide on carbon (Pearlman, Lancaster or Aldrich catalyst, 5.3 g) was added. The mixture was placed in a Parr Pressurized Reactor apparatus with glass insert. The device was placed under nitrogen and then under a hydrogen pressure of 110-120 psi. The mixture was stirred for 2-3 days at room temperature under hydrogen pressure of 100-120 psi. The reaction mixture was placed under nitrogen and filtered through a pad of celite. The celite pad was washed with methanol (100 mL) and water (100 mL). The methanol was removed by roto-evaporation. The aqueous layer was washed with ethyl acetate three times (100, 50, 50 mL).
Juntou-se uma solução de NaOH 10M (10 mL) à camada aquosa (pH = 12-14). O produto foi extraído a partir da camada aquosa três vezes com diclorometano (100, 100, 50 mL) . As camadas orgânicas combinadas foram secas com Na2S04, filtradas e roto-evaporadas até ficar um óleo incolor. O óleo espumoso foi seco sob vácuo durante 2 h. O Intermediário 4 foi obtido num rendimento de 90% (3,34 g).A 10M NaOH solution (10 mL) was added to the aqueous layer (pH = 12-14). The product was extracted from the aqueous layer three times with dichloromethane (100, 100, 50 mL). The combined organic layers were dried over Na2SO4, filtered and rotoevaporated to a colorless oil. The foamed oil was dried under vacuum for 2 h. Intermediate 4 was obtained in 90% yield (3.34 g).
Composto 5 [2-(2',3'-Di-hidro-benzo[1,4]dioxin-6'-il)-2-hidroxi-l-pirrolidin-l-ilmetil-etil]amida de ácido (IR,2R)-hexadecanóico 13 ΕΡ 1 409 467/ΡΤCompound 5 (1R, 2aS, 3aS) -Dihydro-benzo [1,4] dioxin-6'-yl) -2-hydroxy-1-pyrrolidin-1-ylmethyl-ethyl] 2R) -hexadecanoic acid 13 ΕΡ 1 409 467 / ΡΤ
A uma solução de Intermediário 4 (3,34 g, 12,0 mmol) em diclorometano (50 mL) adicionou-se uma solução de N-hidroxi-succinimidoéster de ácido palmítico (Sigma, 4,24 g, 12,0 mmol) durante 15-30 minutos sob azoto a temperatura ambiente. A solução foi agitada a temperatura ambiente durante 18-20 horas. À reacção juntou-se diclorometano (50 mL) e uma solução de NaOH 1M (25 mL) . O sistema de duas fases foi agitado a temperatura ambiente durante 15-30 min. Juntou-se água (25 mL) e as camadas foram separadas. A camada aquosa foi extraída de novo com diclorometano (25 mL) . As camadas orgânicas combinadas foram lavadas duas vezes com água (25 mL) e uma vez com uma solução saturada de cloreto de sódio (25 mL) . A camada orgânica foi seca com Na2S04, filtrada e roto-evaporada até ficar um óleo amarelo claro. O produto bruto foi recristalizado em hexano (50 mL) . O sólido branco (5,46 g) obtido foi separado sobre sílica gel (300 g) com metanol a 2%:diclorometano - metanol a 4%:diclorometano - amónio 2M em metanol a 4%:diclorometano. O sólido branco obtido foi recristalizado em hexanos (70 mL) . O Composto 5 foi obtido num rendimento de 66% (4,18 g) . A HPLC quiral analítica (coluna: Chirex (S)-VAL e (R)-NE, 4,6x250 mm; eluente: ácido trifluoroacético a 0,5% em hexano/dicloro- metano/etanol 67:31:2; caudal: 1 mL/min; detecção: 280 nM) mostrou que este material era 98, 98% puro com 0,89% de um diastereoisómero e 0,14% do enantiómero.To a solution of Intermediate 4 (3.34 g, 12.0 mmol) in dichloromethane (50 mL) was added a solution of palmitic acid N-hydroxysuccinimidoester (Sigma, 4.24 g, 12.0 mmol) for 15-30 minutes under nitrogen at room temperature. The solution was stirred at room temperature for 18-20 hours. To the reaction was added dichloromethane (50 mL) and 1M NaOH solution (25 mL). The two-phase system was stirred at room temperature for 15-30 min. Water (25 mL) was added and the layers were separated. The aqueous layer was extracted again with dichloromethane (25 mL). The combined organic layers were washed twice with water (25 mL) and once with a saturated sodium chloride solution (25 mL). The organic layer was dried over Na2SO4, filtered and rotoevaporated to a pale yellow oil. The crude product was recrystallized from hexane (50 mL). The white solid (5.46 g) obtained was separated on silica gel (300 g) with 2% methanol: 4% dichloromethane-methanol: 2M dichloromethane-ammonium in 4% methanol: dichloromethane. The white solid obtained was recrystallized from hexanes (70 mL). Compound 5 was obtained in 66% yield (4.18 g). Analytical chiral HPLC (column: Chirex (S) -VAL and (R) -NE, 4.6x250 mm; eluent: 0.5% trifluoroacetic acid in 67: 31: 2 hexane / dichloromethane / ethanol: 1 mL / min; detection: 280 nM) showed that this material was 98.98% pure with 0.89% of a diastereoisomer and 0.14% of the enantiomer.
Exemplo comparativo 3 - Preparação alternativa em larga escala de compostos do tipo ceramidaComparative Example 3 - Large-scale alternative preparation of ceramide-type compounds
Sal de HC1 de (5S)-5-fenilmorfolin-2-ona(5 S) -5-Phenylmorpholin-2-one HCl salt
Uma solução de bromoacetato de fenilo (Aldrich, 862,17 g, 4,0 moles, 1,1 equivalentes) em acetonitrilo (qualidade de reagente, 1500 ml) foi arrefecida num banho de gelo (temperatura interna inferior a 5°C). A esta juntou-se uma 14 ΕΡ 1 409 467/ΡΤ lama fria (temperatura interna inferior a 5°C) de S-(+)-2-fenilglicinol (Aldrich, 500 g, 3,65 moles, 1 equivalente) e diisopropiletilamina (DIPEA) (Aldrich, 1587 ml, 9,11 moles, 2,5 equivalentes) em acetonitrilo (2900 ml) em porções, enquanto a temperatura interna era mantida inferior a 10°C. A mistura foi agitada a esta temperatura durante 30 minutos antes do banho de gelo ser removido e a deixou-se a mistura sob agitação a temperatura ambiente adicionalmente durante 4 horas. O solvente foi removido sob vácuo, enquanto se manteve a temperatura do banho a 25°C. A mistura foi co-evaporada com acetato de etilo (2x500 ml) para produzir um óleo viscoso amarelo claro. A esta mistura reaccional juntou-se acetato de etilo (4500 ml) e o frasco foi imerso num banho de gelo com agitação. Deixou-se a mistura arrefecer abaixo de 8°C. O sólido foi filtrado e lavado com acetato de etilo (3x250 ml). A solução foi arrefecida abaixo de 5°C. Foi passado lentamente HC1 gás seco pela solução, enquanto se mantinha a temperatura interna inferior a 15°C, até o pH ser inferior a 2 (papel de pH húmido). Deixou-se a mistura sob agitação a esta temperatura e este pH durante 20 minutos adicionais antes do sólido ser filtrado por sucção. O sólido foi lavado com acetato de etilo (3x200 ml) e seco sob vácuo elevado durante cerca de 20 horas. O rendimento foi de 412 g (53%). O espectro de ΤΗ RMN foi consistente com o sal de HC1 de (5S)-5-fenilmorfolin-2-ona.A solution of phenyl bromoacetate (Aldrich, 862.17 g, 4.0 mol, 1.1 equivalents) in acetonitrile (reagent quality, 1500 ml) was cooled in an ice bath (internal temperature less than 5øC). To this was added a cold slurry (internal temperature less than 5 ° C) of S - (+) - 2-phenylglycinol (Aldrich, 500 g, 3.65 moles, 1 equivalent) and diisopropylethylamine (DIPEA) (Aldrich, 1587 ml, 9.11 moles, 2.5 equivalents) in acetonitrile (2900 ml) was added portionwise while the internal temperature was maintained below 10 ° C. The mixture was stirred at this temperature for 30 minutes before the ice bath was removed and the mixture was left under stirring at room temperature additionally for 4 hours. The solvent was removed under vacuum while maintaining the bath temperature at 25 ° C. The mixture was coevaporated with ethyl acetate (2 x 500 mL) to give a pale yellow viscous oil. To this reaction mixture was added ethyl acetate (4500 mL) and the flask was immersed in an ice bath with stirring. The mixture was allowed to cool below 8 ° C. The solid was filtered and washed with ethyl acetate (3x250 mL). The solution was cooled to below 5 ° C. Dry HCI was slowly passed through the solution while maintaining the internal temperature below 15øC until the pH was less than 2 (wet pH paper). The mixture was allowed to stir at this temperature and this pH for an additional 20 minutes before the solid was filtered by suction. The solid was washed with ethyl acetate (3 x 200 mL) and dried under high vacuum for about 20 hours. The yield was 412 g (53%). The NMR spectrum was consistent with the (5S) -5-phenylmorpholin-2-one HCl salt.
Intermediário 1 (1-R, 35,5S, 8a5)-l,3-Bis-(2',3' -di-hidro-benzo [ 1, 4] dioxin-6' -il)-5-feniltetra-hidro-oxazolo[4,3-c][1,4]oxazin-8-ona A uma suspensão agitada de sal de HC1 de (5S)-5-fenil-morfolin-2-ona (381 g, 1 equivalente) em acetato de etilo a 15% em tolueno (2270 ml) juntou-se uma solução de bicarbonato de sódio (1,1 equivalentes) em água (2000 ml). A solução bifásica resultante foi agitada a temperatura ambiente durante cerca de 1 hora. A camada orgânica foi transferida para um frasco contendo 1,4-benzodioxan-6-carboxaldeido. O frasco foi depois equipado com uma unidade Dean-Stark, um condensador e uma entrada para azoto. A mistura foi aquecida a refluxo com agitação, enquanto cerca de 650 ml de solvente (mistura de acetato de etilo e tolueno) eram recolhidos via unidade de Dean-Stark. A resultante solução amarelo-vermelho foi deixada 15 ΕΡ 1 409 467/ΡΤ sob refluxo durante cerca de 64 horas sob azoto, enquanto a água formada durante a reacção foi recolhida na unidade Dean-Stark. A maior parte do solvente foi depois removida via destilação a pressão atmosférica através da unidade Dean-Stark. 0 solvente residual foi depois removido por co-evaporação com heptano (500 ml) e éter tert-butilmetílico (2x725 ml) para produzir um produto semi-sólido amarelo. O produto semi-sólido foi dissolvido em acetato de etilo (3400 ml). Adicionou-se uma solução de bissulfito de sódio (920 g) em água (1500 ml) e deixou-se a mistura sob agitação a temperatura ambiente durante cerca de 1 hora. O sólido que se formou foi removido por filtração e lavado com acetato de etilo (3x400 ml) . O filtrado foi lavado com água (1450 ml), solução de salmoura a 5% (1450 ml) e seco sobre MgS04 (100 g) . O solvente foi removido sob vácuo para proporcionar um sólido amarelo. A este juntou-se éter tert-butilmetilico (2900 ml) e a suspensão foi agitada a temperatura ambiente durante 20 a 22 horas. O sólido amarelo foi filtrado por sucção, lavado com éter tert-butil-metilico (2x600 ml) e seco sob vácuo elevado a temperatura ambiente durante cerca de 22 horas. O rendimento foi de 400,5 g (58%). Os valores de ΧΗ RMN e TLC eram consistentes com o Intermediário 1.Intermediate 1 (1R, 35,5S, 8aS) -1,3-Bis- (2 ', 3'-dihydro-benzo [1,4] dioxin-6'-yl) -5-phenyltetrahydro oxazolo [4,3-c] [1,4] oxazin-8-one To a stirred suspension of (5S) -5-phenyl-morpholin-2-one HCl salt (381 g, 1 equiv.) in acetate 15% ethyl acetate in toluene (2270 ml) was added a solution of sodium bicarbonate (1.1 equivalents) in water (2000 ml). The resulting biphasic solution was stirred at room temperature for about 1 hour. The organic layer was transferred to a flask containing 1,4-benzodioxan-6-carboxaldehyde. The flask was then fitted with a Dean-Stark unit, a condenser and a nitrogen inlet. The mixture was heated to reflux with stirring while about 650 ml of solvent (mixture of ethyl acetate and toluene) were collected via Dean-Stark unit. The resulting red-yellow solution was allowed to reflux under nitrogen for about 64 hours while the water formed during the reaction was collected in the Dean-Stark unit. Most of the solvent was then removed by distillation at atmospheric pressure through the Dean-Stark unit. The residual solvent was then removed by coevaporation with heptane (500 mL) and tert-butyl methyl ether (2 x 725 mL) to afford a yellow semi-solid. The semi-solid product was dissolved in ethyl acetate (3400 ml). A solution of sodium bisulfite (920 g) in water (1500 ml) was added and the mixture was allowed to stir at room temperature for about 1 hour. The solid which formed was removed by filtration and washed with ethyl acetate (3.times.400 ml). The filtrate was washed with water (1450 ml), 5% brine solution (1450 ml) and dried over MgSO4 (100 g). The solvent was removed in vacuo to provide a yellow solid. To this was added tert-butyl methyl ether (2900 ml) and the suspension was stirred at room temperature for 20 to 22 hours. The yellow solid was suction filtered, washed with tert-butyl methyl ether (2 x 600 mL) and dried under high vacuum at room temperature for about 22 hours. The yield was 400.5 g (58%). The ¹H NMR and TLC values were consistent with Intermediate 1.
Intermediário 2 (25,3-R,l"5)-3-(2',3' -Di-hidro-benzo [1, 4]dioxin-6'-il)-3-hidroxi-2- (2"-hidroxi-l"-fenil-etilamino) -1-pirrolidin-l-il-propan-l-onaIntermediate 2 (25,3-R, 1c) -3- (2 ', 3'-Dihydro-benzo [1,4] dioxin-6'-yl) -3-hydroxy-2- (2 " phenyl) -ethylamino) -1-pyrrolidin-1-yl-propan-1-one
Uma solução do Intermediário 1 (312 g, 0,64 moles), pirrolidina (267 ml, 3,2 moles, 5 equivalentes) e tetra-hidrofurano (1350 ml) foi aquecida a refluxo durante 4,5 horas sob atmosfera de azoto. O solvente e o excesso de pirrolidina foram removidas sob vácuo para produzir o Intermediário bruto como um óleo viscoso laranja. O óleo foi dissolvido em metanol (3000 ml) e uma solução de ácido clorídrico 1M (3000 ml). A solução resultante foi aquecida a refluxo durante cerca de 7 horas. O solvente foi depois removido sob vácuo para proporcionar uma mistura de um óleo e água. A esta juntou-se acetato de etilo (2000 ml) e a camada aquosa foi separada. A camada orgânica foi extraída com HC1 1M aquoso (1000 ml). As camadas aquosas foram combinadas e lavadas com acetato de etilo (2000 ml). A camada aquosa foi arrefecida num banho de 16 ΕΡ 1 409 467/ΡΤ gelo. Ο ρΗ da camada aquosa foi ajustado a cerca de 9 (papel de pH) com NaOH aquoso 10M (525 ml) . A camada aquosa foi extraída com acetato de etilo (3000 ml) . A camada orgânica foi lavada com uma solução de salmoura a 5% (1000 ml) e seca (Na2S04). O solvente foi removido sob vácuo para produzir um óleo viscoso amarelo. O rendimento foi de 213,4 g, 81%. O espectro de ΤΗ RMN era consistente com o Intermediário 2.A solution of Intermediate 1 (312 g, 0.64 moles), pyrrolidine (267 ml, 3.2 mol, 5 equivalents) and tetrahydrofuran (1350 ml) was heated at reflux for 4.5 hours under a nitrogen atmosphere. The solvent and excess pyrrolidine were removed in vacuo to give the crude Intermediate as an orange viscous oil. The oil was dissolved in methanol (3000 ml) and 1M hydrochloric acid solution (3000 ml). The resulting solution was heated at reflux for about 7 hours. The solvent was then removed under vacuum to provide a mixture of an oil and water. To this was added ethyl acetate (2000 ml) and the aqueous layer was separated. The organic layer was extracted with 1M aqueous HCl (1000 ml). The aqueous layers were combined and washed with ethyl acetate (2000 ml). The aqueous layer was cooled in a bath of 16 æl 1 409 467 / æ ice. Ο ρΗ of the aqueous layer was adjusted to about 9 (pH paper) with 10M aqueous NaOH (525 ml). The aqueous layer was extracted with ethyl acetate (3000 ml). The organic layer was washed with a solution of 5% brine (1000 ml) and dried (Na2 SO4). The solvent was removed in vacuo to give a yellow viscous oil. The yield was 213.4 g, 81%. The ¹H NMR spectrum was consistent with Intermediate 2.
Intermediário 3 IR,2R,1"5)-1-(2', 3'-Di-hidro-benzo[1,4]dioxin-6'-il)-2-(2"~ hidroxi-l"-fenil-etilamino)-3-pirrolidin-l-il-propan-l-olIntermediate 3 IR, 2R, 1 " 5) -1- (2 ', 3'-Dihydro-benzo [1,4] dioxin-6'-yl) -2- (2'-hydroxy-1'-phenyl -ethylamino) -3-pyrrolidin-1-yl-propan-1-ol
Adicionou-se a uma lama de LiAlH4 (50,7 g, 1,34 moles, 2,6 equivalentes) em tetra-hidrofurano (700 ml) uma solução de Intermediário 2 (213,34 g, 0,517 moles) em tetra-hidrofurano (2000 ml) lentamente com agitação a temperatura ambiente. A mistura foi levada a refluxo durante cerca de 4 horas. A análise por TLC (metanol a 10% em diclorometano, v/v) indicou o consumo do material de partida. A mistura reaccional foi arrefecida num banho de gelo (inferior a 5°C) e juntou-se água (135 ml) muito lentamente, enquanto era mantida a temperatura interna inferior ou igual a 10°C. A esta juntou-se depois uma solução aquosa de NaOH a 15% (70 ml) seguida de água (200 ml) . Deixou-se a mistura reaccional aquecer até temperatura ambiente enquanto era mantida a agitação. Adicionou-se então diclorometano (1000 ml) à mistura e os sais foram filtrados através de uma almofada de celite. Os sais foram lavados com diclorometano (2x500 ml). Os filtrados foram combinados e o solvente foi removido sob vácuo para produzir um óleo amarelo. O óleo foi dissolvido em HC1 aquoso 1M (1500 ml) e lavado com acetato de etilo (3x500 ml). A camada aquosa foi arrefecida num banho de gelo abaixo de 5°C e o pH da camada aquosa foi ajustado a 12 a 13 com uma solução aquosa de NaOH 10M (220 ml) mantendo a temperatura interna inferior ou igual a 10°C. Deixou-se a mistura aquecer até temperatura ambiente. A camada aquosa foi extraída com diclorometano (2x500 ml) . Combinaram-se as camadas orgânicas e lavaram-se com uma solução de salmoura (500 ml), secas (Na2S04) e o solvente foi removido sob vácuo para proporcionar um óleo amarelo viscoso. O rendimento foi de 186,4 g (88,5%) . O espectro de 2Η RMN era consistente com o Intermediário 3. 17 ΕΡ 1 409 467/ΡΤTo a slurry of LiAlH 4 (50.7 g, 1.34 moles, 2.6 equivalents) in tetrahydrofuran (700 ml) was added a solution of Intermediate 2 (213.34 g, 0.517 mol) in tetrahydrofuran (2000 ml) slowly with stirring at room temperature. The mixture was refluxed for about 4 hours. TLC analysis (10% methanol in dichloromethane, v / v) indicated consumption of the starting material. The reaction mixture was cooled in an ice bath (below 5øC) and water (135 ml) was added very slowly, while maintaining the internal temperature less than or equal to 10øC. To this was then added a 15% aqueous NaOH solution (70 ml) followed by water (200 ml). The reaction mixture was allowed to warm to room temperature while stirring was maintained. Dichloromethane (1000 ml) was then added to the mixture and the salts were filtered through a pad of celite. The salts were washed with dichloromethane (2x500 ml). The filtrates were combined and the solvent was removed in vacuo to give a yellow oil. The oil was dissolved in 1M aqueous HCl (1500 ml) and washed with ethyl acetate (3 x 500 ml). The aqueous layer was cooled in an ice bath to below 5øC and the pH of the aqueous layer was adjusted to 12 to 13 with a 10M aqueous NaOH solution (220 ml) keeping the internal temperature less than or equal to 10øC. The mixture was allowed to warm to room temperature. The aqueous layer was extracted with dichloromethane (2x500 ml). The organic layers were combined and washed with a solution of brine (500 mL), dried (Na2 SO4) and the solvent removed in vacuo to provide a yellow viscous oil. The yield was 186.4 g (88.5%). The 2 H NMR spectrum was consistent with Intermediate 3. 17 Î »1 409 467 / ΡΤ
Intermediário 4, sal dioxalato (IR,2R)-2-Amino-l-(2',3'-di-hidro-benzo[1,4]dioxin-6'-il)-3-pirrolidin-l-il-propan-l-ol, sal dioxalatoIntermediate 4, dioxalate salt (1R, 2R) -2-Amino-1- (2 ', 3'-dihydro-benzo [1,4] dioxin-6'-yl) -3-pyrrolidin- propan-1-ol, dioxalate salt
Uma suspensão de Intermediário 3 (358 g, 0,90 moles), etanol (1500 ml), solução de HC1 1M (1500 ml) e Pd(OH)2 a 10% (32 g, 20% em peso) foi hidrogenada a cerca de 50 psi durante cerca de 36 horas a temperatura ambiente. A mistura foi filtrada através de um filtro Cuono. O filtro Cuono foi lavado com etanol a 10% em água (500 ml). Os filtrados foram combinados e o etanol foi removido sob vácuo. A camada aquosa foi extraída com acetato de etilo (3x600 ml). A camada orgânica foi extraída com HC1 aquoso 1M (700 ml). As camadas aquosas foram combinadas e arrefecidas num banho de gelo (0 a cerca de 5°C). O pH da camada aquosa foi ajustado a cerca de 12 (papel de pH) com uma solução aquosa de NaOH 10M (490 ml) mantendo a temperatura interna inferior a 10°C. Deixou-se a camada aquosa aquecer até temperatura ambiente. A camada aquosa foi extraída com diclorometano (2x1500 ml, 1x750 ml). As camadas orgânicas combinadas foram secas sobre MgS04 e o solvente foi removido sob vácuo para proporcionar um óleo amarelo viscoso. 0 peso bruto foi de 214,3 g (86%). O espectro de ΧΗ RMN era consistente com o Intermediário 4.A suspension of Intermediate 3 (358 g, 0.90 mol), ethanol (1500 ml), 1 M HCl solution (1500 ml) and 10% Pd (OH) 2 (32 g, 20% by weight) was hydrogenated at about 50 psi for about 36 hours at room temperature. The mixture was filtered through a Cuono filter. The Cuono filter was washed with 10% ethanol in water (500 ml). The filtrates were combined and the ethanol was removed in vacuo. The aqueous layer was extracted with ethyl acetate (3x600 ml). The organic layer was extracted with 1M aqueous HCl (700 mL). The aqueous layers were combined and cooled in an ice bath (0 to about 5 ° C). The pH of the aqueous layer was adjusted to about 12 (pH paper) with a 10M aqueous NaOH solution (490 ml) keeping the internal temperature below 10Â ° C. The aqueous layer was allowed to warm to room temperature. The aqueous layer was extracted with dichloromethane (2x1500 ml, 1x750 ml). The combined organic layers were dried over MgSO4 and the solvent was removed in vacuo to provide a viscous yellow oil. The crude weight was 214.3 g (86%). The ¹-NMR spectrum was consistent with Intermediate 4.
Uma solução de ácido oxálico (152,4 g, 1,693 moles, 2,2 equivalentes) em metilisobutilcetona (2300 ml) foi adicionada lentamente com agitação a uma solução de Intermediário 4 (214,3 g, 0,77 moles, 1 equivalente) em metilisobutilcetona (800 ml) a temperatura ambiente. A mistura resultante foi agitada a temperatura ambiente durante cerca de 2,5 horas. O sólido foi filtrado e triturado com acetona (2000 ml) a temperatura ambiente durante cerca de 16 horas. O sólido foi filtrado, lavado com acetona (3x100 ml) e seco sob vácuo elevado para produzir um sólido esbranquiçado. 0 rendimento foi de 312,5 g (89%) . O espectro de RMN era consistente com o sal dioxalato do Intermediário 4.A solution of oxalic acid (152.4 g, 1.693 moles, 2.2 equivalents) in methylisobutylketone (2300 ml) was added slowly with stirring to a solution of Intermediate 4 (214.3 g, 0.77 mol, 1 equivalent) in methylisobutylketone (800 ml) at room temperature. The resulting mixture was stirred at room temperature for about 2.5 hours. The solid was filtered and triturated with acetone (2000 ml) at ambient temperature for about 16 hours. The solid was filtered, washed with acetone (3 x 100 mL) and dried under high vacuum to afford an off-white solid. The yield was 312.5 g (89%). The NMR spectrum was consistent with the dioxalate salt of Intermediate 4.
Composto 5 [2-(2',3'-di-hidrobenzo[1,4]dioxin-6'-il)-2-hidroxi-l-pirrolidin-l-ilmetil-etil]amida de ácido (IR,2R)-hexadecanóico A uma solução fria (cerca de 5°C) de sal dioxalato de Intermediário 4 (507 g, 1,11 moles) em água (10 L) juntou-se 18 ΕΡ 1 409 467/ΡΤ uma solução aquosa de NaOH 10M (500 ml) com agitação, sendo mantida a temperatura interna inferior a 10°C. Deixou-se a solução aquecer até temperatura ambiente, enquanto o pH da solução era mantido a cerca de 14 (papel de pH) . A camada aquosa foi extraída com diclorometano (3x6000 ml). As camadas orgânicas foram combinadas, lavadas com água (2000 ml), secas (MgS04) e o solvente foi removido sob vácuo para proporcionar um óleo viscoso amarelo, Intermediário 4. O rendimento foi de 302 g (98%) . O espectro de ΧΗ RMN era consistente com o Intermediário 4.Compound 5 (1R, 2R) -2-Amino-2- (2 ', 3'-dihydrobenzo [1,4] dioxin-6'-yl) -2-hydroxy-1-pyrrolidin-1-ylmethyl- -hexadecanoic acid To a cold (about 5 ° C) solution of Intermediate 4 dioxalate salt (507 g, 1.11 mol) in water (10 L) was added a 10M aqueous NaOH solution (500 ml) with stirring, the internal temperature being maintained below 10 ° C. The solution was allowed to warm to room temperature, while the pH of the solution was maintained at about 14 (pH paper). The aqueous layer was extracted with dichloromethane (3 x 6000 ml). The organic layers were combined, washed with water (2000 ml), dried (MgSO4) and the solvent removed under vacuum to provide a yellow viscous oil, Intermediate 4. The yield was 302 g (98%). The ¹-NMR spectrum was consistent with Intermediate 4.
Uma solução do NHS-éster de ácido palmítico (Sigma, 382,5 g, 1,01 equivalentes) em diclorometano (2500 ml) foi adicionada a uma solução de Intermediário 4 (302 g) em diclorometano (1500 ml) a temperatura ambiente durante um período de 1,25 horas sob atmosfera de azoto. Deixou-se a mistura sob agitação a temperatura ambiente durante cerca de 18 horas. Juntou-se uma solução aquosa de NaOH 1M (2425 ml) e a mistura foi agitada a temperatura ambiente durante cerca de 3 horas. A camada orgânica foi separada e a camada aquosa foi extraída com diclorometano (800 ml). As camadas orgânicas foram combinadas, lavadas com uma solução de NaOH 1M (3x1500 ml) e água (1500 ml) . A camada orgânica foi seca sobre MgS04 e o solvente foi removido sob vácuo para proporcionar um produto semi-sólido. O semi-sólido foi co-evaporado com heptano (3x100 ml) . O produto bruto foi transferido para um frasco RB de 12 L e três tubuladuras e juntou-se heptano (7500 ml). A mistura foi aquecida a refluxo com agitação sob uma atmosfera de azoto. A solução foi lentamente arrefecida até cerca de 55°C (temperatura interna) e vertida noutro frasco. A solução foi agitada a temperatura ambiente durante 24 horas sob uma atmosfera de azoto. O sólido esbranquiçado foi filtrado, lavado com heptano (2x500 ml) e seco sob vácuo elevado durante 24 horas. O sólido (397 g) foi transferido para um frasco RB de 12 L e juntou-se acetato de etilo a 30% em heptano (8000 ml) . A mistura resultante foi aquecida a refluxo durante 30 minutos com agitação. A solução foi arrefecida a cerca de 55°C (temperatura interna) e vertida noutro frasco. A agitação foi mantida a temperatura ambiente sob uma atmosfera de azoto durante cerca de 24 horas. O sólido foi filtrado, lavado com heptano (2x100 ml) e seco sob vácuo elevado para proporcionar um sólido esbranquiçado. O 19 ΕΡ 1 409 467/ΡΤ rendimento foi de 324 g (58%) . As análises de RMN e TLC foram consistentes com o Composto 5. P.f. 96,1°C. Análise de HPLC: pureza quiral 99,7%, pureza química 99,7%. Anál. calc. para C31H52N2O4: C, 72,05; H, 10,14; N, 5,42; Encontr. C, 72,03; H, 10,19; N, 5,42.A solution of the NHS-ester of palmitic acid (Sigma, 382.5 g, 1.01 equiv) in dichloromethane (2500 ml) was added to a solution of Intermediate 4 (302 g) in dichloromethane (1500 ml) at room temperature for a period of 1.25 hours under a nitrogen atmosphere. The mixture was allowed to stir at room temperature for about 18 hours. Aqueous 1M NaOH solution (2425 ml) was added and the mixture was stirred at room temperature for about 3 hours. The organic layer was separated and the aqueous layer was extracted with dichloromethane (800 ml). The organic layers were combined, washed with 1M NaOH solution (3x1500 ml) and water (1500 ml). The organic layer was dried over MgSO4 and the solvent was removed in vacuo to afford a semi-solid product. The semi-solid was coevaporated with heptane (3x100 ml). The crude product was transferred to a 12 L three-necked RB flask and heptane (7500 ml) was added. The mixture was heated to reflux with stirring under a nitrogen atmosphere. The solution was slowly cooled to about 55øC (internal temperature) and poured into another vial. The solution was stirred at room temperature for 24 hours under a nitrogen atmosphere. The off-white solid was filtered, washed with heptane (2 x 500 ml) and dried under high vacuum for 24 hours. The solid (397 g) was transferred to a 12 L RB flask and 30% ethyl acetate in heptane (8000 ml) was added. The resulting mixture was heated at reflux for 30 minutes with stirring. The solution was cooled to about 55øC (internal temperature) and poured into another vial. Stirring was maintained at room temperature under a nitrogen atmosphere for about 24 hours. The solid was filtered, washed with heptane (2 x 100 mL) and dried under high vacuum to provide an off-white solid. The ε 409 467 / ΡΤ yield was 324 g (58%). NMR and TLC analyzes were consistent with Compound 5. M.p. 96.1 ° C. HPLC analysis: chiral purity 99.7%, chemical purity 99.7%. Anal. calc. for C 31 H 52 N 2 O 4: C, 72.05; H, 10.14; N, 5.42; Found. C, 72.03; H, 10.19; N, 5.42.
Exemplo 4 - Preparação de Compostos 6-8Example 4 - Preparation of Compounds 6-8
Foram preparados ésteres de IV-hidroxi-succinimida de ácidos gordos por meio do processo de Lapidot, Y., Rappoport, S. e Wolman, Y., Journal of Lipid Research 8, 1967, ou como se descreve em seguida: Éster de IV-hidroxi-succinimida de ácido octanóico IV-hidroxi-succinimida (Aldrich, 20,0 g, 173 mmol) e trietilamina (29 mL, 208 mmol) foram dissolvidas em diclorometano num banho de gelo sob azoto. Adicionou-se cloreto de octanoílo (Aldrich, 35 mL, 205 mmol) gota a gota durante 0,5 horas. O banho de gelo foi removido e a solução com um sólido branco foi agitada durante 1 hora a temperatura ambiente. O sólido branco foi removido por filtração e o filtrado foi lavado com água (100 mL) e bicarbonato de sódio aquoso saturado (100 mL) . A camada orgânica foi seca com sulfato de sódio, filtrada e juntou-se heptano (100 mL). A solução foi roto-evaporada para remover a maior parte do diclorometano e deixou um precipitado em heptano, incolor a branco, em flocos. O precipitado foi filtrado e lavado com heptano. Após secagem foi obtido o éster de N-hidroxi-succinimida de ácido octanóico em 84% de rendimento (35,4 g). ΧΗ RMN (CDCI3) 2,84 (br s, 4H) , 2,60 (t, J=7, 48 Hz, 2H) , 1,78- 1,71 (m, 2H), 1,42-1,26 (m, 8H), 0,88 (t, J=6,7 Hz, 3H) ppm.Esters of fatty acid IV-hydroxy succinimide were prepared by the procedure of Lapidot, Y., Rappoport, S. and Wolman, Y., Journal of Lipid Research 8, 1967, or as follows: (Aldrich, 20.0 g, 173 mmol) and triethylamine (29 mL, 208 mmol) were dissolved in dichloromethane in an ice bath under nitrogen. Octanoyl chloride (Aldrich, 35 mL, 205 mmol) was added dropwise over 0.5 hours. The ice bath was removed and the solution with a white solid was stirred for 1 hour at room temperature. The white solid was removed by filtration and the filtrate was washed with water (100 mL) and saturated aqueous sodium bicarbonate (100 mL). The organic layer was dried over sodium sulfate, filtered and heptane (100 mL) was added. The solution was rotoevaporated to remove most of the dichloromethane and left a precipitate in colorless, white, heptane in flakes. The precipitate was filtered and washed with heptane. After drying, the octanoic acid N-hydroxysuccinimide ester was obtained in 84% yield (35.4 g). Δ NMR (CDCl3) 2.84 (br s, 4H), 2.60 (t, J = 7.48 Hz, 2H), 1.78-1.71 (m, 2H), 1.42-1, 26 (m, 8H), 0.88 (t, J = 6.7 Hz, 3H) ppm.
Composto 6 [2-(2',3'-di-hidrobenzo[1,4]dioxin-6'-il)-2-hidroxi-l-pirrolidin-l-ilmetil-etil]amida de ácido (IR,2R)-octanóico 20 ΕΡ 1 409 467/ΡΤ ΟΗCompound 6 (1R, 2R) -2-Amino- (2 ', 3'-dihydrobenzo [1,4] dioxin-6'-yl) -2-hydroxy-1-pyrrolidin-1-ylmethyl- -octanoic acid 20 Ε 1 409 467 / ΡΤ ΟΗ
CH2CI2 ΟΗ N-hidroxi-succinimido-éster de ácido octanóico >CH 2 Cl 2 ΟΗ N-hydroxy succinimido ester of octanoic acid >
(CH2)sCH3 Composto 6(CH 2) s CH 3 Compound 6
Ao Intermediário 4 (22,36 g, 80,33 mmol) dissolvido em diclorometano anidro (300 mL) adicionou-se uma solução de éster de IV-hidroxi-succinimida de ácido octanóico (19,4 g, 80,39 mmol) dissolvido em diclorometano anidro (150 mL) durante 15-30 minutos sob azoto a temperatura ambiente. A solução foi agitada a temperatura ambiente durante 18-20 horas. À reacção foi adicionada uma solução aquosa de NaOH 1M (200 mL) . O sistema de duas fases foi agitado a temperatura ambiente durante 45 minutos. As camadas foram separadas e as camadas orgânicas combinadas foram lavadas duas vezes com NaOH 1M (2x200 mL) e duas vezes com água (2x100 mL) . A camada orgânica foi seca com sulfato de sódio, filtrada e roto-evaporada até ficar um óleo amarelo. A maior parte do material bruto foi dissolvida em acetato de etilo a 5% em heptano (1 L) a refluxo. Após arrefecimento a 40°C, a solução turva foi separada do óleo amarelo por decantação da solução para um novo frasco. 0 primeiro frasco foi enxaguado duas vezes com acetato de etilo a 5% em heptano (2x250 mL) através do mesmo processo (refluxo e arrefecimento a 40 °C e decantação da solução a partir do óleo). A solução combinada foi aquecida a refluxo e deixada arrefecer até temperatura ambiente ao longo de 4 horas. O sólido branco resultante foi filtrado e lavado com acetato de etilo a 5% em heptano (100 mL) e heptano (100 mL). O sólido branco (13,9 g) foi seco sob vácuo durante 16-24 horas. Este sólido foi na maior parte dissolvido em acetato de etilo a 5% em heptano (800 mL) a refluxo. Após arrefecimento a 50°C, a solução turva foi separada do óleo amarelo por decantação da solução para um novo frasco. 0 primeiro frasco foi enxaguado com acetato de etilo a 5% em heptano (100 mL) através do mesmo processo (refluxo e arrefecimento a 50°C e decantação da solução a partir do óleo). A solução combinada foi aquecida a refluxo e deixada arrefecer até temperatura ambiente ao longo de 4 horas. 0 sólido branco resultante foi filtrado e lavado com acetato de etilo a 5%/heptano (50 mL) e 21 ΕΡ 1 409 467/ΡΤ heptano (50 mL). Após secagem a temperatura ambiente sob vácuo durante 2-3 dias, obteve-se o Composto 6 com 39% de rendimento (12,57 g) . Através de HPLC quiral analítica (coluna: Chirex (S)-VAL e (R)-NE, 4,6x250 mm) mostrou-se que este material era 99,9% o desejado isómero R, R. Por HPLC analítica mostrou-se que este material era 99,6% puro. P.f. 87-88°C. 2H RMN(CDC13) δ 6,86-6, 73 (m, 3H) , 5,84 (d, J=7,3 Hz, 1H), 4,91 (d, J=3,4 Hz, 1H), 4,25 (s, 4H), 4,24-4,18 (m, 1H), 2,85-2,75 (m, 2H), 2,69- 2,62 (m, 4H), 2,10 (t, J=7,3 Hz, 2H) , 1,55-1,45 (m, 2H), 1,70- 1,85 (m, 4H), 1,30-1,15 (m, 8H), 0,87 (t, J=6,9 Hz, 3H) ppm.To Intermediate 4 (22.36 g, 80.33 mmol) dissolved in anhydrous dichloromethane (300 mL) was added a solution of octanoic acid IV-hydroxy succinimide ester (19.4 g, 80.39 mmol) dissolved in anhydrous dichloromethane (150 mL) for 15-30 minutes under nitrogen at room temperature. The solution was stirred at room temperature for 18-20 hours. To the reaction was added 1M aqueous NaOH solution (200 mL). The two-phase system was stirred at room temperature for 45 minutes. The layers were separated and the combined organic layers were washed twice with 1M NaOH (2x200 mL) and twice with water (2x100 mL). The organic layer was dried over sodium sulfate, filtered and rotoevaporated to a yellow oil. Most of the crude material was dissolved in 5% ethyl acetate in refluxing heptane (1 L). After cooling to 40øC, the cloudy solution was separated from the yellow oil by decantation of the solution into a new vial. The first flask was rinsed twice with 5% ethyl acetate in heptane (2x250 mL) by the same procedure (refluxing and cooling to 40 ° C and decantation of the solution from the oil). The combined solution was heated to reflux and allowed to cool to ambient temperature over 4 hours. The resulting white solid was filtered and washed with 5% ethyl acetate in heptane (100 mL) and heptane (100 mL). The white solid (13.9 g) was dried under vacuum for 16-24 hours. This solid was mostly dissolved in 5% ethyl acetate in heptane (800 mL) at reflux. After cooling to 50øC, the cloudy solution was separated from the yellow oil by decantation of the solution into a new vial. The first flask was rinsed with 5% ethyl acetate in heptane (100 mL) by the same procedure (refluxing and cooling at 50 ° C and decantation of the solution from the oil). The combined solution was heated to reflux and allowed to cool to ambient temperature over 4 hours. The resulting white solid was filtered and washed with 5% ethyl acetate / heptane (50 mL) and 21 Å-1 409 467 / ΡΤ heptane (50 mL). After drying at room temperature under vacuum for 2-3 days, Compound 6 was obtained in 39% yield (12.57 g). The analytical HPLC was shown to be 99.9% by the analytical chiral HPLC (column: Chirex (S) -VAL and (R) -NE, 4.6x250 mm). that this material was 99.6% pure. Federal Police. 87-88Â ° C. 1 H NMR (CDCl 3) δ 6.86-6.73 (m, 3H), 5.84 (d, J = 7.3 Hz, 1H), 4.91 (d, J = 3.4 Hz, 1H) , 4.25 (s, 4H), 4.24-4.18 (m, 1H), 2.85-2.75 (m, 2H), 2.69-2.62 (m, 4H), 2. , 10 (t, J = 7.3 Hz, 2H), 1.55-1.45 (m, 2H), 1.70-1.85 (m, 4H), 1.30-1.15 , 8H), 0.87 (t, J = 6.9 Hz, 3H) ppm.
Composto comparativo 7 [2-(2' , 3' -di-hidro-benzo[1,4]dioxin-6'-il)-2-hidroxi-l-pirrolidin-l-ilmetil-etil]amida de ácido (IR,2R)-nonanóicoComparative compound 7 (1R, 2 ', 3'-Dihydro-benzo [1,4] dioxin-6'-yl) -2-hydroxy-1-pyrrolidin-1-ylmethyl-ethyl] , 2R) -nonanoic acid
N-hidroxi-succinimido-éster de ácido nonanóico CH2CI2N-hydroxysuccinimido ester of nonanoic acid CH 2 Cl 2
OHOH
Composto 7Compound 7
Este composto foi preparado por meio do processo descrito para o Composto 6, utilizando éster de N-hidroxi-succinimida de ácido nonanóico. Através de HPLC analítica mostrou-se que este material era 98,4% puro. P.f. 74-75°C. 1H RMN (CDC13) δ 6, 86-6, 76 (m, 3H) , 5,83 (d, J= 7,3 Hz, 1H) , 4,90 (d, J= 3,3 Hz, 1H) , 4,24 (s, 4H) , 4,24-4,18 (m, 1H) , 2, 85-2, 75 (m, 2H), 2,69-2,62 (m, 4H), 2,10 (t, J= 7,3 Hz, 2H), 1,55-1,45 (m, 2H) , 1, 70-1,85 (m, 4H) , 1,30-1,15 (m, 10H) , 0,87 (t, J= 6,9 Hz, 3H) ppm.This compound was prepared by the procedure described for Compound 6 using nonanoic acid N-hydroxysuccinimide ester. By analytical HPLC this material was shown to be 98.4% pure. Federal Police. 74-75 ° C. 1 H NMR (CDCl 3) δ 6.86-6.76 (m, 3H), 5.83 (d, J = 7.3 Hz, 1H), 4.90 (d, J = 3.3 Hz, 1H) , 4.24 (s, 4H), 4.24-4.18 (m, 1H), 2.85-2.75 (m, 2H), 2.69-2.62 (m, 4H), 2. , 10 (t, J = 7.3 Hz, 2H), 1.55-1.45 (m, 2H), 1.70-1.85 (m, 4H), 1.30-1.15 , 10H), 0.87 (t, J = 6.9 Hz, 3H) ppm.
Composto comparativo 8 [2-(2',3'-di-hidro-benzo[1,4]dioxin-6'-il)-2-hidroxi-l-pirrolidin-l-ilmetil-etil]amida de ácido (IR,2R)-decanóicoComparative compound 8 (1R) -2- (2 ', 3'-dihydro-benzo [1,4] dioxin-6'-yl) -2-hydroxy-1-pyrrolidin-1-ylmethyl-ethyl] , 2R) -decanoic acid
Composto 8 22 ΕΡ 1 409 467/ΡΤCompound 8 22 Ε 1 409 467 / ΡΤ
Este composto foi preparado por meio do processo descrito para o Composto 6, utilizando éster de N-hidroxi-succinimida de ácido decanóico. Através de HPLC analítica mostrou-se que este material era 99,3% puro. P.f. 97,5-98,5°C. ΤΗ RMN (CDC13) δ 6, 86-6, 76 (m, 3H) , 5,83 (d, J= 7,5 Hz, 1H) , 4,90 (d, J=3,4 Hz, 1H) , 4,24 (s, 4H) , 4,24-4,18 (m, 1H) , 2,85-2,75 (m, 2H), 2,69-2,62 (m, 4H), 2,10 (t, J=7,5 Hz, 2H), 1,55-1,45 (m, 2H) , 1, 70-1,85 (m, 4H) , 1,30-1,15 (m, 12H) , 0,87 (t, J=6,8 Hz, 3H) ppm.This compound was prepared by the procedure described for Compound 6 using decanoic acid N-hydroxysuccinimide ester. By analytical HPLC this material was shown to be 99.3% pure. Federal Police. 97.5-98.5 ° C. Δ NMR (CDCl3) δ 6.86-6.76 (m, 3H), 5.83 (d, J = 7.5 Hz, 1H), 4.90 (d, J = 3.4 Hz, 1H) , 4.24 (s, 4H), 4.24-4.18 (m, 1H), 2.85-2.75 (m, 2H), 2.69-2.62 (m, 4H), 2. , 10 (t, J = 7.5 Hz, 2H), 1.55-1.45 (m, 2H), 1.70-1.85 (m, 4H), 1.30-1.15 , 12H), 0.87 (t, J = 6.8 Hz, 3H) ppm.
Exemplo comparativo 5 - Preparação do Composto 13Comparative Example 5 - Preparation of Compound 13
Intermediário 9 (l-R,35,55,8a5)-l,3-Bis-( 4-benziloxi-f enil) -5-fenil-tetra-hidro-oxazolo[4,3-c][1,4]oxazin-8-onaIntermediate 9 (1R, 35,55,8a, 5) -1,3-Bis- (4-benzyloxy-phenyl) -5-phenyl-tetrahydro-oxazolo [4,3- c] [1,4] oxazin- 8-one
O sal de HC1 de (5S)-5-fenilmorfolin-2-ona (57, 45 g, 268, 9 mmol) foi agitado com acetato de etilo (500 mL) e bicarbonato de sódio aquoso saturado (250 mL) durante 30 minutos, até a solução bifásica se tornar límpida. As fases foram separadas, e a camada aquosa foi extraída com acetato de etilo (2x250 mL). As fases orgânicas combinadas foram lavadas com uma solução saturada de cloreto de sódio (250 mL) . A camada orgânica foi seca com sulfato de sódio, filtrada, concentrada até se obter um óleo, e seca sob vácuo durante 60 minutos. A 5-(S)-fenilmorfolin-2-ona foi obtida num rendimento de 86% (40,98 g, 231,3 mmol). A 5-(S)-fenilmorfolin-2-ona (40,98 g, 231,3 mmol) e 4-benziloxibenzaldeído (Aldrich, 147,3 g, 694 mmol, 3,0 equivalentes) foram dissolvidos em tolueno (750 mL). A reacção foi equipada com um dispositivo de Dean Stark e um condensador de refluxo. A solução foi levada a refluxo sob azoto durante 2 dias. Após arrefecimento até temperatura ambiente, o solvente foi removido por roto-evaporação e o óleo foi dissolvido em acetato de etilo (500 mL). Adicionou-se uma solução de bissulfito de sódio (Aldrich, 125 g) 23 ΕΡ 1 409 467/ΡΤThe (5S) -5-phenylmorpholin-2-one HCl salt (57.45 g, 268.9 mmol) was stirred with ethyl acetate (500 mL) and saturated aqueous sodium bicarbonate (250 mL) for 30 minutes , until the biphasic solution becomes clear. The phases were separated, and the aqueous layer was extracted with ethyl acetate (2x250 mL). The combined organic phases were washed with brine (250 mL). The organic layer was dried over sodium sulfate, filtered, concentrated to an oil, and dried under vacuum for 60 minutes. 5- (S) -phenylmorpholin-2-one was obtained in 86% yield (40.98 g, 231.3 mmol). 5- (S) -phenylmorpholin-2-one (40.98 g, 231.3 mmol) and 4-benzyloxybenzaldehyde (Aldrich, 147.3 g, 694 mmol, 3.0 equivalents) were dissolved in toluene (750 mL ). The reaction was equipped with a Dean Stark device and a reflux condenser. The solution was refluxed under nitrogen for 2 days. After cooling to room temperature, the solvent was removed by rotoevaporation and the oil was dissolved in ethyl acetate (500 mL). A solution of sodium bisulfite (Aldrich, 125 g) was added 23 Ź 1 409 467 / ΡΤ
dissolvido em água (250 mL) e a mistura de duas fases foi agitada a temperatura ambiente durante 3 horas. O sólido branco resultante foi removido por filtração e lavado com acetato de etilo. 0 filtrado foi colocado num funil de separação e as camadas separadas. A camada orgânica foi lavada com água (250 mL), solução aquosa saturada de cloreto de sódio (250 mL) , e depois seca (sulfato de sódio), filtrada e roto-evaporada até um óleo espumoso (144 g) . Após secagem sob vácuo durante 1 hora, juntou-se éter tert-butilmetilico (1450 mL) e a mistura foi agitada a temperatura ambiente durante 5 horas. 0 sólido branco-amarelo resultante foi filtrado. O sólido foi seco sob vácuo. O Intermediário 9 foi obtido num rendimento de 27% (41,64 g, 71,46 mmol) . RMN (CDC13) δ 7,5-6,8 (m, 23H) , 5,0 e 5,1 (2s, 4H) , 4,5-4,3 (m, 2H), 4,2-4,1 (m, 2H) ppm.dissolved in water (250 mL) and the two phase mixture was stirred at room temperature for 3 hours. The resulting white solid was removed by filtration and washed with ethyl acetate. The filtrate was placed on a separatory funnel and the layers separated. The organic layer was washed with water (250 mL), brine (250 mL), and then dried (sodium sulfate), filtered and rotoevaporated to a foam oil (144 g). After drying under vacuum for 1 hour, tert-butylmethyl ether (1450 mL) was added and the mixture was stirred at room temperature for 5 hours. The resulting white-yellow solid was filtered. The solid was dried under vacuum. Intermediate 9 was obtained in 27% yield (41.64 g, 71.46 mmol). NMR (CDCl 3) δ 7.5-6.8 (m, 23H), 5.0 and 5.1 (2s, 4H), 4.5-4.3 (m, 2H), 4.2-4, 1 (m, 2H) ppm.
Intermediário 10 (2S,3R,l"S)-3-( 4-Benziloxi-f enil) -3-hidroxi-2 - (2"-hidroxi-l"-fenil-etilamino)-1-pirrolidin-l-il-propan-l-onaIntermediate 10 (2S, 3R, 1S) -3- (4-Benzyloxy-phenyl) -3-hydroxy-2- (2'-hydroxy-1'-phenyl-ethylamino) -1-pyrrolidin-1-yl -propan-1-one
OHOH
Ao Intermediário 9 (45,1 g, 77,4 mmol) dissolvido em tetra-hidrofurano (250 mL) adicionou-se pirrolidina (Aldrich 33 mL, 395 mmol, 5,1 equivalentes). A solução foi agitada tapada sob azoto a temperatura ambiente durante 16-18 horas. O solvente foi roto-evaporado para proporcionar um óleo amarelo espumoso que foi seco sob vácuo durante 0,5 horas. O produto bruto foi dissolvido em metanol (220 mL) e juntou-se uma solução aquosa de HC1 1M (220 mL). A solução foi levada a refluxo durante 4 horas. Após arrefecimento a temperatura ambiente, o metanol foi removido por roto-evaporação. Ao óleo resultante foi adicionado lentamente NaOH aquoso 10M (22 mL para ajustar o pH a 14) . O produto foi extraído três vezes com diclorometano (300, 100, 100 mL) a partir da camada aquosa básica. Após secagem com sulfato de sódio, a camada orgânica combinada foi filtrada e roto-evaporada para proporcionar um sólido espumoso amarelo-laranja. Juntou-se 24 ΕΡ 1 409 467/ΡΤ éter tert-butilmetílico (300 mL) e a mistura foi agitada a temperatura ambiente durante 7 horas. O sólido branco-amarelo resultante foi filtrado, lavado com éter tert-butilmetilico (50 mL) e seco sob vácuo. O Intermediário 10 foi obtido com rendimento de 83% (29, 77 g) . ΤΗ RMN (CDC13) δ 7,4-7,2 (m, 12H), 6,9-6,8 (m, 2H), 5,05 (quarteto AB, 2H), 4,47 (d, J=8,5, 1H), 3,9-3,3 (m, 3H) , 3,05 (d, J=8,5, 1H) , 3,0-2,8 (m, 2H) , 2,3-2,2 (m, 1H), 1,85-1,7 (m, 1H) , 1,45-1,15 (m, 4H) ppm.To Intermediate 9 (45.1 g, 77.4 mmol) dissolved in tetrahydrofuran (250 mL) was added pyrrolidine (Aldrich 33 mL, 395 mmol, 5.1 equivalents). The solution was stirred under nitrogen at room temperature for 16-18 hours. The solvent was rotoevaporated to provide a foamy yellow oil which was dried under vacuum for 0.5 hours. The crude product was dissolved in methanol (220 mL) and 1M aqueous HCl (220 mL) was added. The solution was refluxed for 4 hours. After cooling to room temperature, the methanol was removed by roto-evaporation. To the resulting oil was slowly added 10M aqueous NaOH (22 mL to adjust the pH to 14). The product was extracted three times with dichloromethane (300, 100, 100 mL) from the basic aqueous layer. After drying with sodium sulfate, the combined organic layer was filtered and roto-evaporated to provide a yellow-orange foamy solid. Tert-butyl methyl ether (300 mL) was added and the mixture was stirred at room temperature for 7 hours. The resulting white-yellow solid was filtered, washed with tert-butyl methyl ether (50 mL) and dried under vacuum. Intermediate 10 was obtained in 83% yield (29.77 g). Δ NMR (CDCl3) δ 7.4-7.2 (m, 12H), 6.9-6.8 (m, 2H), 5.05 (AB quartet, 2H), 4.47 (d, J = 8.5, 1H), 3.9-3.3 (m, 3H), 3.05 (d, J = 8.5, 1H), 3.0-2.8 (m, 2H) 3-2.2 (m, 1H), 1.85-1.7 (m, 1H), 1.45-1.15 (m, 4H) ppm.
Intermediário 11 (lR,2R,l"S)-l-(4-Benziloxi-fenil)-2-(2"-hidroxi-l"-fenil-etilamino)-3-pirrolidin-l-il-propan-l-olIntermediate 11 (1R, 2R, 1S) -1- (4-Benzyloxy-phenyl) -2- (2'-hydroxy-1'-phenyl-ethylamino) -3-pyrrolidin-1-yl-propan- ol
Num frasco de 3 tubuladuras com funil de adição e condensador sob azoto, adicionou-se LiAlH4 (Aldrich, 6,3 g, 166 mmol, 2,57 equivalentes) e tetra-hidrofurano anidro (75 mL) . Juntou-se gota a gota uma solução de Intermediário 10 (29,7 g, 64,48 mmol) em tetra-hidrofurano anidro (300 mL) à reacção durante 15-30 minutos. A reacção foi levada a refluxo sob azoto durante 9 horas. Arrefeceu-se a reacção num banho de gelo e juntou-se água (7,0 mL) muito cuidadosamente e gota a gota (reacção exotérmica vigorosa, com libertação de hidrogénio). Adicionou-se uma solução aquosa de NaOH a 15% (7,0 mL) gota a gota, seguida de água (21 mL) . A meio da adição de água final formou-se uma grande quantidade de um sólido branco. Foi desfeita pela adição de diclorometano (250 mL). Após agitação a temperatura ambiente durante 15 minutos, a mistura foi filtrada através de uma almofada de celite (17 cm de diâmetro por 1 cm de altura). 0 precipitado foi lavado com diclorometano (2x250 mL) . O filtrado foi roto-evaporado até um óleo. O óleo foi dissolvido em HC1 aquoso 1M (300 mL). Esta camada aquosa foi lavada com éter tert-butilmetílico (2x200 mL). Após arrefecimento num banho de gelo, adicionou-se cuidadosamente NaOH aquoso 10M (35 mL) à camada aquosa (pH final = 14) . O produto foi extraído três vezes com 25 ΕΡ 1 409 467/ΡΤ diclorometano (300 mL, 200 mL e 100 mL) . Após secagem com sulfato de sódio, a solução foi filtrada e roto-evaporada para gerar um sólido branco. Após secagem, o Intermediário 11 foi obtido com rendimento de 94% (26,9 g) . ΤΗ RMN (CDCI3) δ 7,46-7,115 (m, 12H), 6,98-6, 96 (m, 2H) , 5,08 (s, 2H) , 4,49 (d, J=4, 7, 1H), 3, 70-3,65 (m, 1H) , 3,60-3,55 (m, 1H), 3,54- 3,45 (m, 1H), 3,00-2,90 (m, 1H), 2,7-2,6 (m, 1H), 2,36 (br s, 4H), 2,15-2,05 (m, 1H), 1,70 (br s, 4H) ppm.In a 3-necked flask with addition funnel and condenser under nitrogen was added LiAlH 4 (Aldrich, 6.3 g, 166 mmol, 2.57 equivalents) and anhydrous tetrahydrofuran (75 mL). A solution of Intermediate 10 (29.7 g, 64.48 mmol) in anhydrous tetrahydrofuran (300 mL) was added dropwise to the reaction over 15-30 minutes. The reaction was refluxed under nitrogen for 9 hours. The reaction was cooled in an ice bath and water (7.0 mL) was added very carefully and dropwise (vigorous exothermic reaction, with hydrogen release). A 15% aqueous NaOH solution (7.0 mL) was added dropwise, followed by water (21 mL). At the middle of the final water addition a large amount of a white solid formed. It was stripped by the addition of dichloromethane (250 mL). After stirring at room temperature for 15 minutes, the mixture was filtered through a pad of celite (17 cm in diameter by 1 cm in height). The precipitate was washed with dichloromethane (2x250 mL). The filtrate was rotoevaporated to an oil. The oil was dissolved in 1M aqueous HCl (300 mL). This aqueous layer was washed with tert-butyl methyl ether (2x200 mL). After cooling in an ice bath, 10M aqueous NaOH (35 mL) was carefully added to the aqueous layer (final pH = 14). The product was extracted three times with 25 æl 1 409 467 / ΡΤ dichloromethane (300 mL, 200 mL and 100 mL). After drying with sodium sulfate, the solution was filtered and roto-evaporated to give a white solid. After drying, Intermediate 11 was obtained in 94% yield (26.9 g). Δ NMR (CDCl3) δ 7.46-7.115 (m, 12H), 6.98-6.96 (m, 2H), 5.08 (s, 2H), 4.49 (d, J = 4.7 , 1H), 3.70-3.65 (m, 1H), 3.60-3.55 (m, 1H), 3.54-3.45 (m, 1H), 3.00-2.90 (m, 1H), 2.7-2.6 (m, 1H), 2.36 (br s, 4H), 2.15-2.05 (m, 1H), 1.70 (br s, 4H ) ppm.
Intermediário 12 (IR,2R)-2-Amino-l-(4-benziloxi-fenil)-3-pirrolidin-l-il-propan-l-ol, sal de cloreto de hidrogénioIntermediate 12 (1R, 2R) -2-Amino-1- (4-benzyloxy-phenyl) -3-pyrrolidin-1-yl-propan-1-ol,
OH -Q.HOH-Q.H
OH O Intermediário 11 (26,9 g, 60,24 mmol) foi dissolvido em metanol (400 mL) e juntou-se HC1 aquoso 1M (130 mL) . Após ter sido colocado sob azoto, adicionou-se hidróxido de paládio sobre carbono a 20% (catalisador de Pearlman, Aldrich, 10,8 g) . A reacção foi colocada sob azoto e depois sob hidrogénio por evacuação e enchimento de um balão. A mistura foi agitada durante 48 horas a temperatura ambiente sob um balão de hidrogénio. A mistura reaccional foi colocada sob azoto e filtrada através de uma almofada de celite. A almofada de celite foi lavada com água a 10% em metanol (250 mL) e água (50 mL) . O solvente foi removido por roto-evaporação e co-evaporação com tolueno (3x100 mL) . O sólido espumoso foi dissolvido em isopropanol (300 mL) a refluxo. A solução foi arrefecida até temperatura ambiente e adicionou-se éter tert-butilmetílico (550 mL). Após agitação a temperatura ambiente durante 2 horas, o sólido branco foi filtrado e lavado com éter tert-butilmetílico. Após secagem, foi obtido o Intermediário 12 com um rendimento de cerca de 99% (18 g). 1H RMN (DMSO-d6) δ 9,68 (br s, 1H) , 8,53 (br s, 2H) 7,24 (d, J= 8,55 Hz, 2H) , 6,80 (d, J= 8,55 Hz, 2H) , 4,72 (d, J=7, 0 Hz, 1H), 3,8-3,6 (m, 2H), 3,4-3,6 (m, 3H), 3,0-3,2 (m, 2H), 2,7-2,5 (br s, 1H), 2,0-1,7 (br s, 4H) ppm. 26 ΕΡ 1 409 467/ΡΤIntermediate 11 (26.9 g, 60.24 mmol) was dissolved in methanol (400 mL) and 1M aqueous HCl (130 mL) was added. After being placed under nitrogen, 20% palladium hydroxide on carbon (Pearlman's catalyst, Aldrich, 10.8 g) was added. The reaction was placed under nitrogen and then under hydrogen by evacuation and filling of a flask. The mixture was stirred for 48 hours at room temperature under a hydrogen flask. The reaction mixture was placed under nitrogen and filtered through a pad of celite. The celite pad was washed with 10% water in methanol (250 mL) and water (50 mL). The solvent was removed by roto-evaporation and coevaporation with toluene (3x100 mL). The foamed solid was dissolved in isopropanol (300 mL) under reflux. The solution was cooled to room temperature and tert-butyl methyl ether (550 mL) was added. After stirring at room temperature for 2 hours, the white solid was filtered and washed with tert-butyl methyl ether. After drying, Intermediate 12 was obtained in about 99% yield (18 g). 1 H NMR (DMSOd 6) δ 9.68 (br s, 1H), 8.53 (br s, 2H) 7.24 (d, J = 8.55 Hz, 2H), 6.80 (d, J = 8.55 Hz, 2H), 4.72 (d, J = 7.0 Hz, 1H), 3.8-3.6 (m, 2H), 3.4-3.6 (m, 3H) , 3.0-3.2 (m, 2H), 2.7-2.5 (br s, 1H), 2.0-1.7 (br s, 4H) ppm. 26 ΕΡ 1 409 467 / ΡΤ
Composto 13 [2-( 4-benziloxi-f enil) -2-hidroxi-l-pirrolidin-l-ilmetil-etiljamida de ácido (IR,2R)-hexadecanóicoCompound 13 (1R, 2R) -hexadecanoic acid [2- (4-benzyloxy-phenyl) -2-hydroxy-1-pyrrolidin-1-ylmethyl-
Ao Intermediário 12 (16,17 g, 49,36 mmol) suspenso em tetra-hidrofurano (500 mL) adicionou-se trietilamina (28 mL, 4 equivalentes). Adicionou-se uma solução de N-hidroxi-succinimidoéster de ácido palmítico (Sigma, 19,2 g, 54,29 mmol) dissolvido em tetra-hidrofurano (125 mL) durante 30 minutos sob azoto a temperatura ambiente. A solução foi agitada a temperatura ambiente durante 18-20 horas. O precipitado branco foi removido por filtração e o filtrado foi roto-evaporado até ser obtido um sólido esbranquiçado espumoso (35,5 g). 0 material bruto foi dissolvido em dicloro-metano (500 mL) e lavado com água (100 mL) e uma solução aquosa saturada de carbonato de sódio (100 mL). Após secagem com sulfato de sódio, a solução foi filtrada e roto-evaporada para gerar um sólido esbranquiçado espumoso (24, 75 g) . Este material foi recristalizado em acetato de etilo a 40% em heptano (500 mL, filtração a quente). O Composto 13 foi obtido com rendimento de 61% (14,45 g) . A HPLC quiral analítica mostrou que este material era 99,7% do desejado isómero R,R. A HPLC analítica mostrou que este material era 99,6% puro. P.f. 95-97°C. 1H RMN (CDC13) δ 7,15 (d, J=8,5Hz, 2H) , 6,70 (d, J= 8,5 Hz, 2H), 6,0 (d, J=7,3, 1H) , 4,96 (d, J=3,8, 1H) , 4,3-4,2 (m, 1H), 2,9-2,7 (m, 2H), 2,65-2,55 (m, 4H), 2,10 (t, J=7,5, 2H), 1,75 (br s, 4H), 1,58-1,46 (m, 2H), 1,32-1,16 (m, 24H) , 0,9 (t, J= 6,7, 3H) ppm.To Intermediate 12 (16.17 g, 49.36 mmol) suspended in tetrahydrofuran (500 mL) was added triethylamine (28 mL, 4 equivalents). A solution of palmitic acid N-hydroxysuccinimidoester (Sigma, 19.2 g, 54.29 mmol) dissolved in tetrahydrofuran (125 mL) was added over 30 minutes under nitrogen at room temperature. The solution was stirred at room temperature for 18-20 hours. The white precipitate was removed by filtration and the filtrate was rotoevaporated to an off-white foamy solid (35.5 g). The crude material was dissolved in dichloromethane (500 mL) and washed with water (100 mL) and a saturated aqueous sodium carbonate solution (100 mL). After drying with sodium sulfate, the solution was filtered and roto-evaporated to give an off-white foamy solid (24, 75 g). This material was recrystallized from 40% ethyl acetate in heptane (500 mL, hot filtration). Compound 13 was obtained in 61% yield (14.45 g). Analytical chiral HPLC showed that this material was 99.7% of the desired R, R isomer. Analytical HPLC showed that this material was 99.6% pure. Federal Police. 95-97 ° C. 1H NMR (CDCl3) δ 7.15 (d, J = 8.5Hz, 2H), 6.70 (d, J = 8.5Hz, 2H), 6.0 (d, J = 7.3, 1H ), 4.96 (d, J = 3.8, 1H), 4.3-4.2 (m, 1H), 2.9-2.7 (m, 2H), 2.65-2.55 (m, 4H), 2.10 (t, J = 7.5, 2H), 1.75 (br s, 4H), 1.58-1.46 (m, 2H) 16 (m, 24H), 0.9 (t, J = 6.7, 3H) ppm.
Lisboa, 2012-07-06Lisbon, 2012-07-06
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